Modulation of cholesteryl ester transfer protein (CETP) activity

ABSTRACT

This invention relates to peptides comprising a helper T cell epitope portion and a B cell epitope portion for eliciting an immune response against endogenous cholesteryl ester transfer protein (CETP) activity, to prevent or treat cardiovascular disease, such as atherosclerosis.

[0001] This is a continuation-in-part application of U.S. patentapplication Ser. No. 08/432,483, filed May 1, 1995.

GENERAL FIELD OF THE INVENTION

[0002] This invention is generally in the field of peptide-basedvaccines to control, treat, or reduce the risk of atherogenic activityin the circulatory system of humans and other animals. In particular,this invention provides compositions and methods for providing means toinhibit the activity of endogenous cholesteryl ester transfer protein(CETP) to treat cardiovascular disease prophylactically or therapeuticalor to modulate the relative levels of lipoproteins to produce acondition correlated with a reduced risk of cardiovascular disease, suchas atherosclerosis.

BACKGROUND OF THE INVENTION

[0003] Cholesterol circulates through the body predominantly ascomponents of lipoprotein particles (lipoproteins), which are composedof a protein portion, called apolipoproteins (Apo) and various lipids,including phospholipids, triglycerides, cholesterol and cholesterylesters. There are ten major classes of apolipoproteins: Apo A-I, ApoA-II, Apo-IV, Apo B-III, Apo B-100, Apo C-I Apo C-II, Apo C-III, Apo D,and Apo E. Lipoproteins are classified by density and composition. Forexample, high density lipoproteins (HDL), one function of which is tomediate transport of cholesterol from peripheral tissues to the liver,have a density usually in the range of approximately 1.063-1.21 g/mL HDLcontain various amounts of Apo A-I, Apo A-II, Apo C-I, Apo C-II, ApoC-III, Apo D, Apo E, as well as various amounts of lipids, such ascholesterol, cholesteryl esters, phospholipids, and triglycerides.

[0004] In contrast to HDL, low density lipoproteins (LDL), whichgenerally have a density of approximately 1.019-1.063 g/mL, contain ApoB-100 in association with various lipids. In particular, the amounts ofthe lipids, cholesterol and cholesteryl esters are considerably higherin LDL than in HDL, when measured as a percentage of dry mass. LDL areparticularly important in delivering cholesterol to peripheral tissues.

[0005] Very low density lipoproteins (VLDL) have a density ofapproximately 0.95-1.006 g/ml and also differ in composition from otherclasses of lipoproteins both in their protein and lipid content. VLDLgenerally have a much higher amount of triglycerides than do HDL or LDLand are particularly important in delivering endogenously synthesizedtriglycerides from liver to adipose and other tissues. The features andfunctions of various lipoproteins have been reviewed (see, for example,Mathews, C. K and van Holde, K. E., Biochemistry, pp. 574576, 626-630(The Benjamin/Cummings Publishing Co., Redwood City, Calif., 1990);Havel R. J., et al., “Introduction: Structure and metabolism of plasmalipoproteins”, In The Metabolic Basis of Inherited Disease, 6th ed., pp.1129-1138 (Scriver, C. R., et al., eds.) (McGraw-Hill, Inc., New York,1989); Zannis, V. I., et al., “Genetic mutations affecting humanlipoproteins, their receptors, and their enzymes”, In Advances in HumanGenetics, Vol, 21, pp. 145-319 (Plenum Press, New York, 1993)).

[0006] Decreased susceptibility to cardiovascular disease, such asatherosclerosis, is generally correlated with increased absolute levelsof circulating HDL and also increased levels of HDL relative tocirculating levels of lower density lipoproteins such as VLDL and LDL(see, e.g., Gordon, D. J., et al., N. Engl. J. Med., 321: 1311-1316(1989); Castelli, W. P., et al, J. Am. Med. Assoc., 256: 2835-2838(1986); Miller, N. E., et al., Am. Heart J., 113: 589-597 (1987); TallA. R., J. Clin. Invest., 89: 379-384 (1990); Tall, A. R., J. InternalMed., 237: 5-12 (1995)).

[0007] Cholesteryl ester transport protein (CETP) mediates the transferof cholesteryl esters from HDL to TG-rich lipoproteins such as VLDL andIDL, and also the reciprocal exchange of TG from VLDL to HDL (Tall, A.R., J. Internal Med., 237: 5-12 (1995); Tall A. R., J. Lipid Res., 34:1255-1274 (1993); Hesler, C. B., et al., J. Biol. Chem., 262:2275-2282(1987); Quig, D. W. et al., Ann. Rev. Nutr., 10: 169-193(1990)). CETP may play a role in modulating the levels of cholesterylesters and triglyceride associated with various classes of lipoproteins.A high CETP cholesteryl ester transfer act has been correlated withincreased levels of LDL-associated cholesterol and VLDL-associatedcholesterol, which in turn are correlated with increased risk ofcardiovascular disease (see, e.g., Tato, F., et al., Arterioscler.Thromb. Vascular Biol., 15: 112-120(1995)).

[0008] Hereinafter, LDL-C will be used to refer to total cholesterol,including cholesteryl esters and/or unesterified cholesterol associatedwith low density lipoprotein. VLDL-C will be used to refer to totalcholesterol including cholesteryl esters and/or unesterified cholesterolassociated with very low density lipoprotein. HDL-C will be used torefer to total cholesterol, including cholesteryl esters and/orunesterified cholesterol, associated with high density lipoprotein.

[0009] CETP isolated from human plasma is a hydrophobic glycoproteinhaving 476 amino acids and a molecular weight of approximately 66,000 to74,000 daltons on sodium dodecyl sulfite (SDS)polyacrylamide gels(Albers, J. J., et al, Arteriosclerosis, 4: 49-58 (1984); Hesler, C. B.,et al., J. Biol. Chem., 262: 2275-2282 (1987); Jarnagin, S. S., et al.,Proc. Natl. Acad. Sci. USA, 84: 1854-1857 (1987)). A cDNA encoding humanCETP has been cloned and sequenced (Drayna, D., et al., Nature, 327:632-634 (1987)). Polymorphism in human CETP has recently been reportedand may be associated with disease in lipid metabolism (Fumeron et al.,J. Clin. Invest., 96: 1664-1671 (1995); Juvonen et al., J. Lipid Res.,36: 804-812 (1995)). CETP has been shown to bind CE, TG, phospholipids(Barter, P. J. et al., J. Lipid Res., 21:238-249 (1980)), andlipoproteins (see, e.g., Swenson, T. L., et al., J. Biol. Chem., 264:14318-14326 (1989)). More recently, the region of CETP defined by thecarboxyl terminal 26 amino acids, and in particular amino acids 470 to475, has been shown to be especially important for neutral lipid bindinginvolved in neutral lipid transfer (Hesler, C. B., et al., J. Biol.Chem., 263: 5020-5023 (1988)), but not phospholipid binding (see, Wang,S., et al., J. Biol. Chem., 267: 17487-17490 (1992); Wang, S., et al.,J. Biol. Chem., 270: 612-618 (1995)).

[0010] A monoclonal antibody (Mab), TP2 (formerly designated 5C7 in theliterature), has been produced which inhibits completely the cholesterylester and triglyceride transfer activity of CETP, and to a lesser extentthe phospholipid transfer activity (Hesler, C. B., et al, J. Biol.Chem., 263: 5020-5023 (1988)). The epitope of TP2 was localized to thecarboxyl terminal 26 amino acids, i.e.,. the amino acids fromargnine-451 to serine-476, of the 74,000 dalton human CETP molecule(see, Hesler, C. B., et al, (1988)). TP2 was reported to inhibit bothhuman and rabbit CETP activity in vitro and rabbit CETP in vivo (Yen, F.T., et al., J. Clin Invest., 83: 2018-2024 (1989) (TP2 reacting withhuman CETP); Whitlock et al., J. Clin. Invest., 84: 129-137 (1989) (TP2reacting with rabbit CETP)). Further analysis of the region of CETPbound by TP2 revealed that amino acids between phenylalanine-463 andleucine-475 are necessary for TP2 binding and for neutral lipid (e.g.,cholesteryl ester) transfer activity (see, Wang, S., et al., 1992).

[0011] A number of in vivo studies utilizing animal models or humanshave indicated that CETP activity can affect the level of circulatingcholesterol-containing HDL. Increased CETP cholesteryl ester transferactivity can produce a decrease in HDL-C levels relative to LDL-C and/orVLDL-C levels which in turn is correlated with an increasedsusceptibility to atherosclerosis. Injection of partially purified humanCETP into rats (which normally lack CETP activity), resulted in a shiftof cholesteryl ester from HDL to VLDL, consistent with CETP-promotedtransfer of cholesteryl ester from HDL to VLDL (Ha, Y. C., et al.,Biochim. Biophys. Acta, 833: 203-211 (1985); Ha, Y. C., et al., Comp.Biochem. Physiol., 83B: 463-466 (1986); Gavish D., et al., J. LipidRes., 28: 257-267 (1987)). Transgenic mice expressing human CETP werereported to exhibit a significant decrease in the level of cholesterolassociated with HDL (see, e.g., Hayek, T., et al., J. Clin. Invest., 90:505-510 (1992); Breslow, J. L., et al., Proc. Natl. Acad Sci. USA, 90:8314-8318 (1993)). Furthermore, whereas wild-type mice are normallyhighly resistant to atherosclerosis (Breslow, J. L., et al., Proc. Natl.Acad Sci. USA, 90: 8314-8318 (1993)), transgenic mice expressing asimian CETP were reported to have an altered distribution of cholesterolassociated with lipoproteins, namely, elevated levels of LDL-C andVLDL-C and decreased levels of HDL-C (Marotti K. R., et al., Nature,364: 73-75 (1993)). Transgenic mice expressing simian CETP also weremore susceptible to dietary-induced severe atherosclerosis compared tonon-expressing control mice and developed atherosclerotic lesions intheir aortas significantly larger in area than those found in thecontrol animals and having a large, focal appearance more typical ofthose found in atherosclerotic lesions in humans (Marotti et al., id.).Intravenous infusion of anti-human CETP monoclonal antibodies (Mab) intohamsters and rabbits inhibited CETP activity in vivo and resulted insignificantly increased levels of HDL-C levels, decreased levels ofHDL-triglyceride, and increased HDL size; again implicating a criticalrole for CETP in the distribution of cholesterol in circulatinglipoproteins (Gaynor, B. J., et al., Atherosclerosis, 110: 101-109(1994) (hamsters); Whitlock, M. E., et al., J. Clin. Invest., 84:129-137 (1989) (rabbits)).

[0012] CETP deficiency has also been studied in humans. For example, incertain familial studies in Japan, siblings that were homozygous fornon-functional alleles of the CETP gene had no detectable CETP activity.Virtually no atherosclerotic plaques were exhibited by theseindividuals, who also showed a trend toward longevity in their families(see, e.g., Brown, M. L., et al., Nature, 342: 448-451 (1989); Inazu, A,et al., N. Engl. J. Med., 323: 1234-1238 ( 990); Bisgaier, C. L., etal., J. Lipid Res., 32: 21-23 (1991)). Such homozygous CETP-deficientindividuals also were shown to have an anti-atherogenic lipoproteinprofile as evidenced by elevated levels of circulating HDL rich incholesteryl ester, as well as overall elevated levels of HDL, andexceptionally large HDL, i.e., up to four to six times the size ofnormal HDL (Brown, M. L., et al., 1989, p. 451). The frequency of thismutation in Japan is relatively high, and may account for an elevatedlevel of HDL in a significant fraction of the Japanese population.

[0013] The above studies indicate that CETP plays a major role intransferring cholesteryl ester from HDL to VLDL and LDL, and thereby inaltering the relative profile of circulating lipoproteins to one whichis associated with an increased risk of cardiovascular disease (e.g.,decreased levels of HDL-C and increased levels of VLDL-C and LDL-C).Marotti et al. (above) interpreted their data as indicating that aCETP-induced alteration in cholesterol distribution was the principalreason that arterial lesions developed more rapidly in transgenic,CETP-expressing mice than in non-transgenic control mice when bothgroups were fed an atherogenic diet. Taken together, the currentevidence suggests that increased levels of CETP activity may bepredictive of increased risk of cardiovascular disease. Modulation orinhibition of endogenous CETP activity is thus an attractive therapeuticmethod for modulating the relative levels of lipoproteins to reduce orprevent the progression of; or to induce regression of; cardiovasculardiseases, such as atherosclerosis.

[0014] It would be advantageous, therefore, to discover compounds andmethods to control CETP activity which would be helpful in preventing ortreating cardiovascular disease. To be an effective pharmacologicaltherapeutic, a compound when administered to a significant majority ofrecipients, ideally, would not elicit an immune response whichneutralizes the beneficial activity or effect of the therapeuticcompound, must not promote a hypersensitive state in the individualreceiving the therapeutic compound, and must not produce untoward sideeffects. It would also be advantageous if such compounds and methodsavoided the necessity for continuous or frequently repeated treatments.

SUMMARY OF THE INVENTION

[0015] This invention provides compounds and methods useful for themodulation or inhibition of cholesteryl ester transfer protein (CETP)activity. In particular, vaccine peptides are described which, whenadministered to a mammal raise an antibody response against the mammal'sown endogenous CETP thereby promoting a prophylactic or therapeuticeffect against cardiovascular disease, such as atherosclerosis. Suchvaccine peptides comprise a helper T cell epitope portion comprising a“universal” or “broad range” immunogenic helper T cell epitope, linked,preferably covalently, to a B cell epitope portion comprising one ormore B cell epitopes from CETP, such as found in the carboxyl terminalportion of human CETP protein that is involved in a neutral lipidbinding or a transfer activity of CETP. Other B cell epitopes from CETPmay also be used. Preferably, the B cell epitopes from CETP used in theB cell epitope portion of the vaccine peptides of this invention induceantibodies to endogenous CETP (autoreactive antibodies) which eitherblock CETP function or lead to clearance of circulating CETP in theblood. In addition, the B cell epitopes used in the vaccine peptides ofthis invention preferably do not also comprise a T cell epitope of CETPso that the possibility of T cell-mediated autoimmune liver damage isavoided.

[0016] The vaccine peptides of this invention include various“multivalent” embodiments. For example, multivalent peptides present theimmune system with more than a single universal or broad range helper Tcell or B cell epitope. Such multivalent vaccine peptides include thosewhich have multiple (two or more) copies of the same or differentuniversal or broad range immunogenic helper T cell epitope and/ormultiple copies of the same or different B cell epitope from the CETPprotein. Those peptides having more than one unique B cell epitope towhich different antibodies can bind may promote the formation of immunecomplexes to effectively clear CETP from the circulatory system.

[0017] In a preferred embodiment, the helper T cell epitope portion of avaccine peptide of this invention is derived from an amino acid sequenceof a universally (broad range) immunogenic helper T cell epitope, suchas those found in tetanus and diptheria toxoids, or in antigenicpeptides known from pertussis vaccine, Bacile Calmette-Guerin (BCG),polio vaccine, measles vaccine, mumps vaccine, rubella vaccine, purifiedprotein derivative (PPD) of tuberculin, Mycobacterium tuberculosishsp-70, and keyhole limpet hemocyanin. Furthermore, various universal(or broad range) antigenic helper T cell epitopes may be linked to oneanother to form multiple (i.e., multivalent) universal antigenic helperT cell epitope portions of the vaccine peptides of this invention.

[0018] In a more preferred embodiment of a vaccine peptide of thisinvention, an amino terminal cysteine residue is covalently linked to anamino acid sequence of a broad range or universal antigenic helper Tcell epitope of tetanus toxoid forming the sequence C Q Y I K A N S K FI G I T E (amino acids 1 to 15 of SEQ ID NO:2), which is covalentlylinked to a B cell epitope portion of a vaccine peptide having thecarboxyl terminal CETP amino acid sequence F G F P E H L L V D F L Q S LS (amino acids 16 to 3 of SEQ ID NO:2).

[0019] In another preferred embodiment, a multivalent vaccine peptidecomprises an amino acid sequence of a broad range or universal antigenichelper T cell epitope of tetanus toxoid, which in turn is covalentlylinked to a B cell epitope portion consisting of two B cell epitopes ofCETP. In one such preferred embodiment of this invention, themultivalent vaccine peptide has the amino acid sequence of SEQ ID NO:8:C Q Y I K A N S K F I G I T E L F P R P D Q Q H S V A Y T F E E D I F GF P E H L L V D F L Q S L S in which an amino terminal cysteine islinked to a T cell epitope from tetanus toxoid (amino acids 2 to 15 ofSEQ ID NO:8) linked to an amino acid sequence containing two B cellepitopes of human CETP, i.e., amino acids 349 to 367 and amino acids 461to 476 of the amino acid sequence for mature human CETP (SEQ ID NO:4).In still another preferred embodiment of this invention, a multivalentvaccine peptide of this invention contains B cell epitopes from thehomologous regions of the rabbit CETP (i.e., amino acids 350 to 368 and481 to 496 of SEQ ID NO:6) and has the amino acid sequence of SEQ IDNO:9:M Q Y I K A N S K F I G I T E R F P R P D G R E A V A Y R F E E D IF G F P K H L L V D F L Q S L S, in which an amino terminal methionineis linked to a T cell epitope from tetanus toxoid (amino acids 2 to 15of SEQ ID NO: 8) which is linked to an amino acid sequence containingthe two B cell epitopes from rabbit CETP.

[0020] The peptides of this invention may also be linked to one anothervia a bifunctional linker molecule or a peptide linker molecule havingminimal or no immunogenicity. In addition, the peptides may be linked toa common molecule to form peptide assemblies in which multiple copies ofthe peptides are arranged close to one another. Such multicopy(multivalent) peptide assemblies may be more immunogenic, that is,produce a more effective immune response to endogenous CETP thanvaccines comprising unassociated individual peptides.

[0021] The vaccine compounds of this invention also may be used incombination with a pharmaceutically acceptable adjuvant.

[0022] The immunogenic vaccine peptides of this invention elicit theproduction of antibodies that are reactive with or recognize endogenousCETP. Administration of vaccine peptides to test animals resulted in adecline in the relative levels of total cholesterol and HDL-C andresulted in a decrease in the development of atherosclerotic lesions.The elicited endogenous anti-CETP antibodies thus promote aphysiological condition correlated with decreased risk of cardiovasculardisease, and they appear to have a direct effect on preventing ordecreasing the formation of atherosclerosis plaques.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1. Flow chart of the protocol for administration of a vaccinepeptide to (vaccination of) rabbits and for withdrawing blood samplesfor analysis of vaccine efficacy. A control rabbit received no vaccinepeptide.

[0024]FIG. 2. Optical density (O.D.) at 450 nm versus plasma dilutionbased on ELISA for anti-CETP antibody binding to recombinant CETP indiluted plasma samples taken from rabbits (rb#1-#4) on Day 70. Opensquare (rabbit rb#4) refers to the plasma of a rabbit not administeredthe vaccine peptide (control). Solid square, circle, and diamond referto rabbits rb#1, #2, and #3, respectively, which were administered avaccine peptide having the amino acid sequence of SEQ ID NO:2.

[0025]FIG. 3. Optical density (O.D.) at 450 nm versus plasma dilutionfor blood plasma samples from rabbits (rb#1-#4, see description of FIG.2, above) based on competitive ELISA for inhibition of monoclonalantibody (Mab) TP2 binding to recombinant human CETP by anti-CETPantibody in diluted rabbit blood plasma samples taken on Day 70.

[0026]FIG. 4. Concentration of total cholesterol (mg/dl) in plasmasamples of rabbits (rb#l -#4, see description of FIG. 2, above) versustime (Days) in vaccination protocol.

[0027]FIG. 5. Concentration of HDL-C (mg/dl) in plasma samples ofrabbits (rb#1-#4, see description of FIG. 2, above) versus time (Days)in vaccination protocol

[0028]FIG. 6. Ratio Non-HDL/HDL in New Zealand white rabbis on Day 70.Control non-vaccinated rabbit (N=1) rb#4 (solid bar); average (N=3) ofvaccinated rabbits rb# 1, 2, and 3 (hatched).

[0029]FIG. 7. Optical density (O.D.) at 450 nm versus plasma dilution(semi-logarithmic graph) based on ELISA for anti-CETP antibody bindingto recombinant CETP in diluted plasma samples taken from humanCETP-transgenic mice on Day 70 in the vaccination protocol. The data foreach mouse administered a vaccine peptide having the amino acid sequenceof SEQ ID NC:2 is indicated by “+” and a solid line. Data for eachcontrol mouse is indicated by “x” and a dashed line. Plasma dilutionsspanned a range of 1:10 to 1:1,000,000 (1E+1 to 1E+6).

[0030]FIGS. 8A and 8B. Typical plots of Hydrophilicity, SurfaceProbability, Antigenic Index, and Amphilic Helix (FIG. 8A) andAmphiphilic Sheets and Secondary Structure (FIG. 8B) for mature humanCETP.

[0031]FIG. 9. Antibody titer to recombinant human CETP from Groups I andII of Atherosclerosis Model based on ELISA. OD at 405 nm versus rabbitplasma dilution.

[0032]FIGS. 10A and 10B. Rabbit plasma antibody titers to rabbit CETP inAtherosclerosis Model based on ELISA. Pre-vaccination plasma (FIG. 10A).Post-vaccination plasma (FIG. 10B). OD at 405 nm versus rabbit plasmadilution.

[0033]FIG. 11. Total cholesterol in pre- and post-vaccinated animals inAtherosclerosis Model. Total cholesterol (mg/dl) versus day and group.

[0034]FIG. 12. HDL-C in pre- and post-vaccinated animals inAtherosclerosis Model. HDL-C (mg/dl) versus day and group.

[0035]FIG. 13. Bar graphs of the average percent of total area of aortacovered by atherosclerotic lesions in vaccinated and control rabbits oncholesterol supplemented diets. Individual data points (diamonds),average percent of aortic area covered by lesions (shaded bar), standarddeviation of data points (open bar), p<0.01 indicates statisticalsignificance between bar graphs.

DETAILED DESCRIPTION OF THE INVENTION

[0036] As noted above, a decreased risk of atherosclerosis has beencorrelated with relatively low circulating levels of LDL and VLDL andrelatively high levels of HDL. The levels of such circulatinglipoproteins are directly influenced, at least in part, by theendogenous levels of CETP activity. In particular, high CETP activitypromotes transfer of neutral lipids, such as cholesteryl esters from HDLto VLDL and IDL. Accordingly, CETP is a relatively precise target inhumans and other animals for altering the relative levels of LDL, VLDLand HDL in the circulatory system (see, e.g., Tato, F., et al.,Arteriosclero. Thromb. Vascular Biol., 15: 112-120(1995); Tall, A R, J.Internal Med., 237: 5-12 (1995)). This invention is directed to thecontrol of endogenous CETP activity by providing CETP vaccine peptidesuseful for promoting an immune response in individuals against theirendogenous CETP, thereby promoting a physiological condition, e.g.,increased level of HDL or decreased level of LDL, correlated with adecreased risk of atherosclerosis. In addition, promoting an immuneresponse against endogenous CETP using the vaccine peptides of thisinvention can provide, prevent, or inhibit the progression of lesions intissue susceptible to atherosclerosis.

[0037] 1. Peptides and Compositions for Modulation of CETP Activity

[0038] As used herein, a CETP vaccine peptide is a peptide comprising ahelper T cell epitope portion comprising an amino acid sequence of auniversal (i.e., broad range) antigenic helper T cell epitope and a Bcell epitope portion comprising an amino acid sequence of a B cellepitope of CETP, such as the carboxyl terminal region of CETP involvedin neutral lipid binding and/or neutral lipid transfer activity. SuchCETP vaccine peptides are antigenic, that is, they elicit production ofspecific antibodies for that peptide which bind endogenous CETP. Thus,the CETP vaccine peptides of this invention are immunogenic moietiesthat have the capacity to stimulate the formation of antibodies whichspecifically bind endogenous CETP and/or inhibit endogenous CETPactivity.

[0039] A. Helper T Cell Epitope Portion of Vaccine Peptides

[0040] Peptides useful in the compositions and methods of this inventioncomprise a helper T cell epitope portion and a B cell epitope portion.The helper T cell epitope portion (or simply, “T cell epitope portion”)has an amino acid sequence derived from at least one universal antigenic(or universal immunogenic or broad range) helper T cell epitope (alsocalled an immunogenic carrier peptide), which is defined as a peptide,or derivative thereof, which can be presented by multiple majorhistocompatibility complex (MHC) haplotypes and thereby activate helperT cells, which in turn, stimulate B cell growth and differentiation. Asdiscussed further below, the B cell epitope portion (also called aCETP-related peptide portion) of the vaccine peptides described hereinhas an amino acid sequence comprising a B cell epitope of the CETPprotein, such as a portion of the carboxyl terminal region of the enzymeCETP that is involved in neutral lipid binding and/or neutral lipidtransfer.

[0041] Examples of what are termed “universal” or “broad range”antigenic helper T cell epitopes which have been used as immunogeniccarrier peptides for human vaccination are known in the art. Theseinclude, for example, epitopes of tetanus toxoid (tt) and diptheriatoxoid (dt) (see, e.g., Panina-Bordignon, P., et al., Eur. J. Immunol.,19: 2237-2242 (1989) (characterization of universal tetanus toxoidhelper T cell epitope peptides); Etlinger, H, Immunol. Today, 13: 52-55(1992); Valmori, D., et al., J. Immunol, 149: 717-721 (1992) (use ofuniversal tt epitopes in candidate anti-malarial vaccine); Talwar, G.P., et al., Proc. Natl. Acad. Sci. USA, 91: 8532-8536 (1994) (use of ttand dt as universal epitopes in anti-human chorionic gonadotropinvaccine); Talwar, G. P., et al., Proc. Natl. Acad. Sci. USA, 91:8532-8536 (1994)). In addition to tt and dt, other helper T cell epitopesequences useful in this invention include those derived from antigenicpeptides known from pertussis vaccine, Bacile Calmette-Guerin (BCG),polio vaccine, measles vaccine, mumps vaccine, rubella vaccine, andpurified protein derivative (PPD) of tuberculin (see, e.g., Etlinger,H., Immunol. Today, 13: 52-55 (1992)); incorporated herein byreference). Furthermore, two or more copies of the same or variousdifferent universal antigenic helper T cell epitopes may be linked toone another to form multiple or multivalent helper T cell epitopeportions of the vaccine peptides of this invention. For example, avaccine peptide of this invention can be synthesized containing amultiple or multivalent helper T cell epitope portion comprising anamino acid sequence of a tt helper T cell epitope and a dt helper T cellepitope.

[0042] In addition, immunogenicity of a vaccine peptide of thisinvention may be further enhanced by linking the helper T cell epitopeportion to a peptide sequence of a xenogeneic CETP or a related proteinhomologous to CETP. Such an approach was used previously in a humanvaccine to human chorionic gonadotropin (see, Talwar, G. P., et al.,Proc. Natl. Acad Sci. USA., 91: 8532-8536 (1994)); heterospecies dimerformed between an amino acid sequence from β subunit of human chorionicgonadotropin and an amino acid sequence of α subunit of ovineluteinizing hormone). Examples of proteins related to CETP that might beused in this approach include, for example, phospholipid transferprotein and neutrophil bactericidal protein (see, Day, J. R., et al., J.Biol. Chem., 269: 9388-91(1994); Gray, P. W., et al., J. Biol. Chem.,264: 9505-9509 (1989)).

[0043] Other immunogenic carrier molecules such as keyhole limpethemocyanin (KLH) may also be used alone or in combination with otheruniversal antigenic helper T cell epitopes. For example, KLH containsmultiple lysine residues in its amino acid sequence. Each of theselysines is a potential site at which a vaccine peptide described hereincould be linked (for example, maleimide-activated KLH, Catalog No.77106, Pierce;,Rockford, Ill.). Such an arrangement is a vaccine peptideassembly that is extensively multivalent for both helper T cell epitopes(i.e., those helper T cell epitopes of the KLH amino acid sequence incombination with those helper T cell epitopes of the multiple copies ofthe attached vaccine peptides) and B cell epitopes of CETP (i.e., thoseB cell epitopes of CETP in the multiple copies of the vaccine peptidesattached to the KLH amino acid sequence).

[0044] Recently, another immunogenic carrier molecule, hsp70 fromMycobacterium tuberculosis, has been shown to be an especially potentantigen containing multiple B and T cell epitopes (Suzue and Young, J.Immunol., 156: 873-879 (1996)). The hsp70protein can be linked bystandard cross-linking agents to vaccine peptides of this invention toenhance immunogenicity. Alternatively, nucleic acid molecules coding forvaccine peptides of this invention can be inserted into an expressionvector which permits the expression of a recombinant protein consistingof the vaccine peptide fused to the amino terminus of hsp70.

[0045] Preferably, the helper T cell epitope portion of the vaccinepeptides of this invention comprises a universal antigenic tt or dthelper T cell epitope. In a more preferred embodiment, the peptides ofthis application use universal antigenic tt helper T cell epitopeshaving amino acid sequences Q Y I K A N S K F I G I T E (amino acids 2to 15 of SEQ ID NO:2)and F N N F T V S F W L R V P K V S A S H L E (SEQID NO:3). Most preferably, the peptides of this invention use theuniversal antigenic tt helper T cell epitope having the amino acidsequence Q Y I K A N S K F I G I T E (amino acids 2 to 15 of SEQ IDNO:2).

[0046] In addition to the various examples of helper T cell epitopesdiscussed above, whether another peptide or protein is useful as ahelper T cell epitope for the T cell epitope portion of the vaccinepeptides of this invention can be determined using a standardproliferation assay for class II (helper) T cell epitopes (see, forexample, pages 3.12.9-3.12.14, In Current Protocols in Immunology, Vol.1 (Coligan et al., eds.) (John Wiley & Sons, Inc., New York, N.Y.,1994)).

[0047] B. B Cell Epitope (CETP-Related) Portion of Vaccine Peptides

[0048] The B cell epitope portion of the vaccine peptides describedherein comprise one or more B cell epitopes of the CETP proteinendogenous to the vaccinated mammal or one or more B cell epitopes of aCETP different from the endogenous CETP but which is immunologically(antibody) cross reactive with the endogenous CETP.

[0049] The B cell epitope portion of the vaccine peptides of thisinvention may comprise one or more B cell epitopes of the endogenousCETP of the individual to be vaccinated for raising antibodies thatinhibit the endogenous CETP activity. However, it is also within thescope of this invention that the B cell epitope portion of the vaccinepeptides of this invention comprise B cell epitopes of CETP moleculesthat are similar, but not identical, to the endogenous CETP of theindividual to be vaccinated. Certain B cell epitopes of such similar,but non-identical, CETP proteins may contain epitopes which enhance theimmune response in the vaccinated individual Generally, CETP moleculeswhich have amino acid sequences that are at least approximately 80percent homologous to the endogenous CETP may be used as a source of Bcell epitopes in the B cell epitope portion of the vaccine peptides ofthis invention. As an example, the rabbit and human CETP proteins havean amino acid sequence homology of approximately 80 percent. The maturerabbit CETP has the amino acid sequence of SEQ ID NO:6 and the maturehuman CETP from liver has the amino acid sequence of SEQ ID NO:4.Accordingly, in an example of such an embodiment of the vaccine peptidesof this invention, the B cell epitope portion comprises one or more Bcell epitopes of a rabbit and/or a human CETP, and such a vaccinepeptide may be used in either rabbits or humans to inhibit theendogenous CETP activity.

[0050] In addition, the B cell epitope portion of the vaccine peptidesof this invention comprises a portion of the amino acid sequence of themature CETP protein (SEQ ID NO: 4) consisting of at least 6 amino acidsequences in length and which does not significantly, if at all,stimulate T cell proliferation in vitro.

[0051] In a preferred embodiment, the vaccine peptides of this inventionhave multivalent B cell epitope portions of vaccine peptides of thisinvention which comprise two or more different B cell epitopes of CETP.Such mulivalent helper T cell epitope portions are especially preferredbecause they present multiple target sites at which elicited antibodiescan bind to the endogenous CETP thereby promoting more extensive immunecomplex formation and/or the likelihood of inhibiting CETP cholesteryltransfer activity.

[0052] In addition, it is preferred that a B cell epitope portion of thevaccine peptides should not comprise a B cell epitope which alsocomprises a T cell epitope that can be presented by endogenous MHC classI molecules. Such T cell epitopes of CETP could be presented on thesurface of hepatocytes a the context of MHC class I and elicit acytotoxic T cell response and thereby damage liver tissue. Whether aparticular CETP B cell epitope comprises a class I T cell epitope can bedetermined using a standard cytotoxic T cell assay (see, for example,pages 3.11.4-3.11.7, In Current Protocols in Immunology Vol. 1 (JohnWiley & Sons, Inc., New York, N.Y., 1994)).

[0053] In another embodiment, the B cell epitope portion of the vaccinepeptides of this invention comprises the carboxyl terminal 26 aminoacids of human CETP (see SEQ ID NO: 1) or fragments thereof that retaina conformation or an activity of the carboxyl terminal 26 amino acidregion of CETP, e.g., fragments of the CETP carboxyl terminus which areat least six consecutive amino acids in length and which are involved inspecific neutral lipid binding and/or specific neutral lipid transferactivity of CETP. More preferably, the B cell epitope (or CETP-related)portion of the vaccine peptides of this invention comprises any fragmentof the carboxyl terminal region of CETP which is at least elevenconsecutive amino acids in length, which retains the conformation of thecarboxyl terminal 26 amino acid region of CETP, which is involved in theneutral lipid binding and/or transfer activity of CETP, and which isaccessible to antibody binding (see, e.g., Wang, S., et al., J. Biol.Chem., 267: 17487-17490 (1992); Wang, S., et al., J. Biol. Chem., 268:1955-1959 (1993)). In addition, several Mabs have been generated to thisregion, including the Mab TP2, that block function of human CETP andalso rabbit CETP, implying that this epitope is conserved by human andrabbit CETP proteins (Whitlock et al., J. Clin. Invest., 84: 129-137(1989). This is confirmed by the fact that the carboxyl terminal 22amino acids of human and rabbit CETP differ at only one position, i.e.,the glutamic acid residue at position 465 in the amino acid sequence ofhuman CETP in SEQ ID NO: 4 is replaced with a lysine at the homologousposition (position 485) in the rabbit sequence (SEQ ID NO:6; see also,Nagashima et al., J. Lipid Res., 29: 1643-1649 (1988)).

[0054] Alternatively, the B cell epitope portion comprises a derivativeof the carboxyl terminal 26 amino acid region of CETP containing aminoacid changes (deletions, additions or substitutions) that do notsignificantly alter or destroy the neutral lipid binding or transferactvity of CETP (see, Wang et al., id, (1992); Wang et al., id, (1993)).Such changes in the amino acid sequence of a targeted endogenous CETPinclude, but are not limited to, what are generally known asconservative amino acid substitutions, such as subs g an amino acid ofthe CETP sequence with another of similar structure, charge, orhydrophobicity. Any addition or substitution to the CETP sequence thatmaintains neutral lipid binding and/or transfer activity, but improvesstability in vivo or in situ, improves purification, or providescross-lining sites (e.g., via cysteine-cysteine disulfide bondformation) is also useful in the design of a vaccine peptide of thisinvention.

[0055] Because CETP-mediated transfer of neutral lipids necessarilyrequires binding of the neutral lipid (e.g., triglycerides, cholesterylester), portions of the amino acid sequence of CETP that are involved inneutral lipid binding are also useful in designing the vaccine peptidesof this invention. Some portions of the amino acid sequence of CETP usedto design the vaccine peptides of this invention may be involved in bothneutral lipid binding as well as the actual catalytic neutral lipidtransfer site of CETP. Recent evidence suggests that CETP containsseparate binding sites for cholesteryl ester and triglycerides(Melchior, G. W., et al., J. Biol. Chem., 270: 21068-21074 (1995)).Accordingly, incorporating the amino acid sequence for a specific lipidbinding site into a vaccine peptide may provide a means to modulate CETPinteractions with that specific lipid thereby promoting ananti-atherogenic profile even though the elicited antibodies to CETP donot promote clearance (reduce serum half-life) of circulating CETP. Forinstance, to modulate triglyceride content of HDL specifically, a B cellepitope derived from the trigylceride binding region of CETP could beincorporated into the B cell epitope portion of a vaccine peptide ofthis invention. Similarly, to modulate cholesteryl ester content of HDLspecifically, a B cell epitope derived from the cholesteryl esterbinding region of CETP could be incorporated into the design of avaccine peptide of this invention. Such vaccine peptides are thusdesigned to elicit antibodies which block specific lipid binding siteson CETP and thereby influence the specific lipid transferred between HDLand CETP.

[0056] Also useful are amino acid sequences of CETP that are at leastsix consecutive amino acids in length, a minimal size of an epitope in aprotein (see, e.g., Watson et al., Molecular Biology of the Gene, 4thedition, page 836 (The Benjamin/Cummings Publising Co., Inc., MenloPark, Calif., 1987), and more preferably, that are at least elevenconsecutive amino acids in length of the carboxyl terminal 26 amino acidregion of CETP encoded by any naturally occurring polymorphisms of theCETP gene.

[0057] CETP molecules of known amino acid sequence can be analyzed forthe location of potential B cell epitopes using algorithms which canidentify potential antigenic motifs in the amino acid sequence. Forexample, by combining analyses of plots of hydrophilicity, surfaceprobability, amphilic helix, amphiphilic sheets and secondary structure(FIGS. 8A and 8B), an Antigenic Index (see FIG. 8A) of the entireprotein's amino acid sequence can be derived leading to identificationof B cell epitopes potentially useful in the vaccine peptides of thisinvention.

[0058] Methods for testing CETP molecules for neutral lipid binding ortheir effect on neutral lipid transfer activity are well known in theart, (see, e.g., Swenson, T. L., et al., J. Biol. Chem., 263: 5150-5157(1988) (assay for lipid binding); Hesler, C. B., et al., J. Biol. Chem.,262: 2275-2282 (1987) (assay for lipid transfer); Bisgaier, C. L, etal., J. Lipid Res., 34: 1625-1634 (1993) (use of fluorescent cholesterylester microemulsions in CETP-mediated cholesteryl transfer activityassay); Gaynor, B. J., et al., Atherosclerosis, 110: 101-109 (1994)(assay for CETP lipid transfer); Wang et al. (1992) (assaying deletionmutants of CETP for transfer activity); Wang et al., (1993) (assayingsingle amino acid mutant forms of CETP); incorporated herein byreference). Assays for the transfer activity of CETP are alsocommercially available (e.g., CETP functional assay by DiagnescentTechnologies, Yonkers, N.Y.).

[0059] Preferably, the B cell epitope portion of the CETP vaccinepeptides of this invention comprises the amino acid sequence the aminoacid sequence L F P R P D Q Q H S V A Y T F E E D I (amino acids 16 to34 of SEQ ID NO: 8) and/or the amino acid sequence F G F P E H L L V D FL Q S L S (amino acids 35 to 50 of SEQ ID NO:8).

[0060] C. Production of Vaccine Peptides

[0061] The helper T cell epitope and the B cell epitope (CETP-related)portions of the CETP vaccine peptides of this invention are linkedtogether to form immunogenic moieties. The helper T cell epitope and Bcell epitope portions may be covalently linked, directly (e.g., via apeptide bond) or via a cross-linking molecule. Where cross-linkingmolecules are used, they must join the helper T cell epitope and B cellepitope portions of the vaccine peptide together without making thepeptide toxic or significantly interfering with or reducing the overallimmunogenicity of the vaccine peptide. Suitable cross-linking moleculesinclude amino acids, for example, using one or more glycine residues toform a “glycine bridge” between the helper T cell epitope and B cellepitope portions of the vaccine peptides of this invention. Alsocontemplated are the formation of disulfide bonds between cysteineresidues that have been added to the helper T cell epitope and B cellepitope portions, or the use of cross-linking molecules such asglutaraldehyde (Korn, A. H., et al., J. Mol. Biol., 65: 525-529 (1972))and EDC (Pierce, Rockford, Ill.) or other bifunctional cross-linkermolecules to link a helper T cell epitope portion to a B cell epitopeportion. Bifunctional cross-linker molecules possess two distinctreactive sites; one of the reactive sites can be reacted with afunctional group on the helper T cell epitope portion to form a covalentlinkage and the other reactive site can be reacted with a functionalgroup on a B cell epitope portion to form another covalent linkage,uniting the two portions. General methods for crosslinking molecules arereviewed by Means and Feeney (Bioconjugate Chem., 1: 2-12 (1990)).

[0062] Homobifunctional cross-linker molecules have two reactive siteswhich are chemically the same. Examples of homobifunctional cross-linkermolecules include glutaraldehyde;N,N′-bis(3-maleimido-propionyl)2-hydroxy-1,3-propanediol (asulfhydryl-specific homobifunctional cross-linker); certainN-succinimide esters, such as disucciimidyl suberate anddithio-bis-(succinnmidyl propionate) and their soluble bis-sulfonicacids and salts (e.g., as available from Pierce Chemicals, Rockford,Ill.; Sigma Chemical Co., St. Louis, Mo.). For this embodiment, therelative concentrations of helper T cell epitope and B cell epitopeportions should be adjusted to maximize the number of helper T cellepitope and B cell epitope portions that are linked together and tominimize the linking of identical epitope portions to each other (i.e.,to avoid, for example, helper T cell epitope-helper T cell epitope or Bcell epitope-B cell epitope homodimer formation).

[0063] Preferably, the bifunctional cross-linker molecule is aheterobifunctional linker molecule, meaning that the linker molecule hasat least two reactive sites that can be separately covalently attachedto a T cell epitope and a B cell epitope. Use of such heterobifunctionallinker molecules permits chemically separate and stepwise addition(vectorial conjugation) of each of the reactive sites of the linkermolecule to the helper T cell and B cell epitope portions of the vaccinepeptide. Heterobifunctional cross-linker molecules that may be used tolink helper T cell epitope and B cell epitope portions together otherinclude m-maleimidobenzoyl-N-hydroxysuccnimide ester (see, Green, N., etal., Cell, 28: 477-487 (1982); Palker et al., Proc. Natl. Acad Sci. USA,84: 2479-2483 (1987); m-maleimido-benzoylsulfosuccinimide ester;γ-maleimidobutyric acid N-hydroxysuccinimide ester; and N-succinimidyl3-(2-pyridyl-dithio)propionate (see, Carlsson, J., et al., Biochem. J.,173: 723-737 (1978); Sigma Chemical Co., St. Louis, Mo.).

[0064] Furthermore, the helper T cell epitope and B cell epitopeportions may be linked to a common carrier molecule, such as serumalbumin or a resin or polymeric bead. Linking helper T cell epitope andB cell epitope portions to a common carrier may be accomplished using across-linker molecule such as glutaraldehyde or other bifunctionalcross-linker molecule (see above). For this embodiment, the relativeconcentrations of helper T cell epitope portion, B cell epitope portionand the common carrier molecule should be adjusted to maximize thenumber of helper T cell epitope and B cell epitope portions that arelinked to the common carrier and to minimize both the linking ofidentical molecules to each other (i.e., homodimer formation) and theling of helper T cell epitope and B cell epitope portions to one another(i.e., heterodimer formation). Linking the helper T cell epitope and Bcell epitope portions to another molecule or surface (e.g., the surfaceof a resin or polymer bead) should not significantly disrupt or reducethe immunogenic characteristics of the universal antigenic helper T cellepitope portion or of the B cell epitope (CETP-related) portionsequences. The net effect of using such bifunctional cross-linkermolecules is that multiple copies of helper T cell epitope and B cellepitope portions of a vaccine peptide are bound to a common carrierwhich may enhance an immune response and the production of antibodiesthat bind to endogenous CETP.

[0065] Multiple antigenic peptide (MAP) arrangements have also beendemonstrated to be highly effective antigens and immunogens (see, e.g.,Tam, J. P., Proc. Natl. Acad. Sci. USA, 85: 5409-5413 (1988); Wang, C.Y., et al., Science, 254: 285-288 (1991); Marguerite, M., et al., Mol.Immunol., 29: 793-800 (1992)). Such MAP technology, in which the helperT cell epitope and B cell epitope portions of the vaccine peptidesdescribed herein are attached to a common core molecule, providesanother way to make multivalent peptide assemblies to elicit antibodiesagainst endogenous CETP.

[0066] Preferably, the helper T cell epitope and B cell epitope portionsof the vaccine peptides of this invention are covalently linkedend-to-end to form a continuous peptide. Most preferably, a selecteduniversal antigenic helper T cell epitope portion forms the aminoterminal portion of the vaccine peptide with its carboxyl terminal aminoacid residue covalently linked in a peptide bond to the amino terminalamino acid of a selected CETP-related amino acid sequence (B cellepitope portion) of the vaccine peptide. However, the reverse order mayalso be used, i.e., the CETP-related amino acid sequence (B cell epitopeportion) of the vaccine peptides of this invention may be positioned toform the amino terminal region of a vaccine peptide and a universalantigenic helper T cell epitope or immunogenic carrier amino acidsequence may comprise the carboxyl terminal portion of the vaccinepeptide.

[0067] The vaccine peptides of this invention can be made moreimmunogenic by covalently linking them to multiple copies of thecomplement protein C3d (Dempsey et al., Science, 271: 348-350 (1996)).Alternatively, the vaccine peptides can be derivatized with carbohydratestructures which activate complement and become covalently linked withC3d (Fearon et al., Science, 272: 5054 (1996)). For example, proteinsexpressed in certain mutant Chinese hamster ovary host cells can beglycosylated with specific carbohydrate structures (Stanley, Mol. Cell.Biol., 9:377-383 (1989)). Recent evidence demonstrates that C3d promotesthe recognition of antigens by the acquired immune system elicitingvigorous immune response (Dempsey et al., 1996).

[0068] The peptides of this invention can be produced by any of theavailable methods known in the art to produce peptides of defined aminoacid sequence. For example, automated peptide synthesis is available tothose skilled in the art by using automated peptide synthesizers (e.g.,Synergy™ Peptide Synthesizer by Applied Biosystems; AMS 422 by Abimed,Langenfeld, Germany). Synthesis of such peptides to order is performedas a commercial service by many commercial peptide synthesizing servicecompanies, e.g., Quality Controlled Biochemicals, Inc., Hopkinton,Mass.); Chiron Mimotopes Peptide Systems (San Diego, Calif.); BachemBioscience, Inc. (Philadelphia, Pa.); Severn Biotech Ltd. (Kiddeminster,England).

[0069] Alternatively, the peptides of this invention may be producedusing synthetic and recombinant nucleic acid technology. For example,one of ordinary skill in the art can design from the known genetic codea 5′ to 3′ nucleic acid sequence encoding a peptide of this invention.The amino acid sequence for a mature CETP from human liver is known (SEQID NO:4), as is its corresponding DNA sequence (SEQ ID NO:5) (see,Drayna et al., Nature, 327: 632-634 (1987)). Furthermore, the amino acidsequences for a variety of broad range or “universal” T cell epitopesare known (see, for example, Panina-Bordignon et al., Eur. J. Immunol.,19: 2232-2242 (1989), Etlinger et al. (1990), Pillai et al., Infect.Immun., 63: 1535-1540 (1995)).

[0070] A DNA molecule containing the coding sequences of a helper T cellepitope and one or more selected B cell epitope portions (and anylinking peptide, such as polyglycine, or other additional residue(s),such as an amino and/or carboxyl terminal cysteine, if so desired) canreadily be synthesized either using an automated DNA synthesizer (e.g.,Oligo 1000 DNA Synthesizer, Beckman Corp.) or by contracting with acommercial DNA synthesizing service (e.g., Genset Corp., La Jolla,Calif.). The synthesized DNA molecule can then be inserted into any of avariety of available gene expression systems (e.g., bacterial plasmids;bacteriophage expression vectors, retroviral expression vectors,baculoviral expression vectors), using standard methods available in theart (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual Vols.1-3 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989))and/or as directed by the manufacturer of a particular commerciallyavailable gene expression system (e.g., pPROEX™-1 bacterial cellexpression system; SFV eukaryotic cell expression system; BAC-TO-BAC™baculovirus expression system; Life Technologies, Inc., Gaithersburg,Md.). The expressed peptide is then isolated from the expression systemusing standard methods to purify peptides. Purification of the peptidesof this invention may be expedited by employing affinity chromatographyor immunoprecipitation based on using antibodies to the particularhelper T cell epitope or B cell epitope (CETP-related portion) aminoacid sequence of a vaccine peptide of this invention. For example, theMab TP2 binds to the carboxyl terminal 26 amino acids of human CETP, andcould be useful in one or more immunoaffiniity steps in a purificationscheme (Hesler, C. B., et al., J. Biol. Chem., 263: 5020-5023 (1988)).

[0071] Of course, if DNA molecules are available encoding each of the Tcell and B cell epitopes for a particular vaccine peptide, standardrecombinant nucleic acid methodologies, including polymerase chainreaction (PCR), can be employed to produce recombinant nucleic acidmolecules encoding the vaccine peptides. Such recombinant nucleic acidmolecules can be inserted into any of a variety of expression vectorswhich can be transfected or transformed into appropriate host cells toexpress the vaccine peptide in culture (see, e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual Vols. 1-3 (Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989)). For example, the DNAsequence encoding the mature human CETP from liver has the nucleotidesequence of SEQ ID NO:5 and the DNA sequence encoding the mature rabbitCETP has the nucleotide sequence of SEQ ID NO:7. Particular sequencesencoding various B cell epitopes of each of these CETP proteins can berecombined with nucleotide sequences encoding selected T cell epitope(s)and inserted into an expression vector for expression in an appropriatehost cell.

[0072] An example of a recombinant plasmid that can be used to produce avaccine peptide is plasmid pCMV-CETP/TT in which the CMV promoterdirects transcription of a sequence encoding a vaccine peptide havingthe amino acid sequence of SEQ ID NO:9: M Q Y I K A N S K F I G I T E RF P R P D G R E A V A Y R F E E D I F G F P K H L L V D F L Q S L S,wherein an amino terminal methionine is linked to a tetanus toxoid Tcell epitope (amino acids 2 to 15 of SEQ ID NO:2) and two B cellepitopes from rabbit CETP (amino acids 350 to 368 and 481 to 496 of SEQID NO:6). Plasmid pCMV-CETP/TT has been deposited with the American TypeCulture Collection (ATCC, Rockville, Md.) and assigned Accession No.98038.

[0073] In a preferred embodiment, a peptide of this invention alsocontains an amino terminal cysteine residue, or other residue,covalently linked to the amino terminal amino acid of the helper T cellepitope portion of the vaccine peptide of this invention for use intethering or coupling the peptide to itself to form dimers of vaccinepeptides or to other molecules, such as carrier or crosslinkermolecules. More preferably, the vaccine peptide of this invention hasthe amino acid sequence C Q Y I K A N S K F I G I T E F G F P E H L L VD F L Q S L S (SEQ ID NO:2). Even more preferred is a vaccine peptide ofthis invention having at least two B cell epitopes of CETP. An exampleof such a vaccine peptide has the sequence C Q Y I K A N S K F I G I T LF P R P D Q Q H S V A Y T F E E D I F G F P E H L L V D F L Q S L S (SEQID NO:8).

[0074] D. Production of Vaccine

[0075] The peptides of this invention are used to make vaccines thatelicit production of endogenous antibodies which specifically bind toCETP and/or modulate (i.e., decrease or inhibit) endogenous CETPactivity. The anti-CETP vaccines of this invention may contain one orseveral different peptides of this invention. For example, peptideshaving different helper T cell epitope portions (e.g., differentuniversal helper T cell epitopes) and/or different B cell epitopeportions (e.g., different CETP-related portions of the carboxyl terminal26 amino acids of CETP) may be combined and administered as a singlevaccine composition.

[0076] Pharmaceutically acceptable adjuvants, such as alum, may be mixedwith vaccine peptides described herein to produce vaccines of thisinvention. Alum is the single adjuvant currently approved for use inadministering vaccines to humans (see, Eldrige, J. R, et al., InImmunobiology of Proteins and Peptides V: Vaccines: Mechanisms, Design,and Applications, Atassi M. Z., ed. (Plenum Press, New York, 1989), page192). Recently, alum was used in combination with a sodium phthalylderivative of lipopolysaccharide to administer a vaccine shown to beeffective against human chorionic gonadotropin to humans (see, Talwar,G. P., et al., Proc. Natl. Acad. Sci. USA, 91: 8532-8536 (1994)).

[0077] Other conventional adjuvants may be used as they are approved fora particular use. For example, biodegradable microspheres comprised ofpoly (DL-lactide-co-glycolide) have been studied as adjuvants for oralor parenteral administration of vaccines (Eldridge, J. H., et al., InImmunobiology of Proteins and Peptides V: Vaccines: Mechanisms, Design,and Applications, Atassi, M. Z., ed. (Plenum Press, New York, 1989), pp.191-202).

[0078] Other adjuvants have been used for administering vaccines tonon-human mammals. For example, Freund's Complete Adjuvant (SigmaChemical Co., St. Louis, Mo.), Freund's Incomplete Adjuvant (SigmaChemical Co., St. Louis, Mo.), and the RIBI™ Adjuvant System (RAS; RIBIImmunoChem Research, Inc., Hamilton, Mont.) are well known adjuvantsroutinely used to administer antigens to animals other than humans. Inaddition, adjuvant structures may also be mixed with or, preferably,covalently incorporated into peptides of this invention, for example atthe amino or carboxyl terminal amino acid residue of the peptides. Suchincorporated adjuvants include lipophilicN-palmitoyl-S-[2,3-bis(palmitoyloxy)-propyl)]-cysteine (“Pam₃-Cys-OH”)at the amino terminus of the peptides of this invention; amphiphilic,water-soluble lipopeptides such as Pam₃-Cys-Ser-Lys₄ andPam₃-Cys-Ser-Glu₄; glycopeptides such asN-acetyl-glucosaminyl-N-acetylmuramyl-alanyl-D-isoglutamine (“GMDP”),muramyl dipeptides, and alanyl-N-adamantyl-D-glutamine; and polyamidegel-based adjuvants which can easily be attached to peptides duringtheir in vitro chemical synthesis (see, Synthetic Vaccines, Nicholson,B. H., ed. (Blackwell Scientific Publications, Cambridge, Mass., 1994),pp. 236-238).

[0079] In addition, the vaccine peptides of this invention may be linkedto other molecules that may enhance the immunogenicity of the peptides.For example, linking peptides of this invention to a surface of a largermolecule, such as serum albumin, may enhance immunogenicity because theepitopes of the vaccine peptides are presented to the immune system ofan individual as adjacent multiple repeated copies (see, e.g., Tam, J.P., Proc. Natl. Acad. Sci. USA, 85: 5409-5413 (1988); Wang, C. Y., etal., Science, 254: 285-288 (1991); Marguerite, M., et al., Mol.Immunol., 29: 793-800 (1992)). Such “multiple” or “multivalent”arrangements of the vaccine peptides of this invention can be createdusing cross-linker molecules (see above). For example, as noted above,bifunctional cross-linker molecules possess two reactive sites, one ofthe sites can attach the linker to a vaccine peptide of this inventionand the other site is available to react with a different molecule,e.g., a larger protein like serum albumin or a resin or polymeric bead.Thus, covalent cross-linker molecules may be used to link vaccinepeptides to other proteins or substrates to form multicopy arrangementsof the peptides (multicopy peptide assemblies).

[0080] Lining vaccine peptides of this invention to another molecule orsurface should be carried out in a manner that does not significantlydisrupt or reduce the immunogenic characteristics of the linked helper Tcell epitope and B cell epitope (CETP-related) portions of the vaccinepeptides. Preferably, the use of such linker molecules enhances theimmunogenicity of the vaccine peptides of this invention as evidenced,for example, by a more rapid rise in anti-CETP antibody titer and/orproduction of higher affinity anti-CETP antibodies than when individualsare administered vaccine peptides that are not linked. Such cross-linkermolecules may also be used to attach a peptide of this invention to an“immunogenic enhancer” molecule such as granulocyte-macrophagecolony-stimulating factor (GM-CSF), which was been shown to serve as aneffective immunogenic enhancer in generating the production of specificanti-tumor antibodies (e.g., Tao, M. H, et al., Nature, 362: 755-758(1993)). Another such immunogenic enhancer is keyhole limpet hemocyanin(KLH) (see, Ada, G. L., In Fundamental Immunology, third edition, W. E.Paul ed. (Raven Press Ltd., New York, 1993), pp. 1309-1352). As notedabove, an example of a mulivalent arrangement using KLH is theattachment of vaccine peptides to any of several cysteine residues ofKLH molecule via disulfide bond formation.

[0081] 2. Use of Vaccine Peptides

[0082] General methods of administering and testing vaccines are wellknown to those skilled in the art (see, e.g., Talwar, G. P., et al.,Proc. Natl. Acad Sci. USA, 91: 8532-8536 (1994)). The peptides of thisinvention are specifically designed to be administered, either alone orin association with one or more pharmaceutically acceptable carriers oradjuvants, as a vaccine which will elicit an antibody response againstendogenous CETP of the vaccine recipient. In some embodiments of thisinvention, the vaccine peptides may also be combined and administeredwith vaccines for other diseases or disorders.

[0083] The immune response to endogenous CETP should significantlyinhibit CETP activity, alter the serum half-life of CETP, causeclearance CETP through formation of immune complexes, alter thetrafficking of HDL-cholesterol shift the equilibrium of cholesterolcontent of lipoproteins, alter cholesterol catabolism, and/or reducedevelopment of atherosclerotic lesions. Control of LDL, VLDL and/or HDLlevels is an accepted indicator or endpoint in treatment ofcardiovasclar disease as these levels are correlated with a decreasedrisk of cardiovascular disease or further progression of such disease(Mader, S. S., In Human Biology. 4th ed., pp. 83, 102 (Wm. C. BrownPublishers, Dubuque, Iowa, 1995)). Accordingly, the desired prophylacticor therapeutic effect according to this invention is evidenced byeliciting antibodies in an individual that bind to CETP and/or inhibitCETP activity, or by a relative decrease in LDL and/or VLDL levelscompared to HDL levels as the titer of antibody directed against theendogenous CETP rises, or by an elevation of absolute levels ofcirculating HDL with the production of anti-CETP antibodies, or by aninhibition or decrease in development of atherosclerotic lesions incardiovascular issue.

[0084] As demonstrated herein, administration of vaccine peptides in arabbit model of atherosclerosis led to a significant decrease in thedevelopment of atherosclerotic plaques in animals fed a cholesterolsupplemented diet. This evidence indicates that vaccination to elicitantibodies to endogenous CETP may be a useful method of treating orpreventing atherosclerosis. This is the first evidence that eliciting animmune response to CETP can inhibit the development of atherosclerosis.

[0085] Such endogenously produced antibodies against an individual's ownCETP is advantageous over other possible therapeutic approaches. Forexample, use of polypeptide inhibitors of CETP, such as one recentlyisolated from baboons (see, e.g., WO 93/11782; Kushwaha, R. S., et al.,J. Lipid Res., 34: 1285-1297 (1993); Genetic Engineering News, 14: 44(1994); Science, 262: 1974-1975 (1993)), or infusion of exogenouslyproduced (foreign) anti-CETP-antibodies which inhibit CETP activity, areboth likely to elicit an immune reaction directed against such foreignCETP inhibitory molecules. Such an immune response could rapidlyinactivate and/or clear from the body the exogenously supplied CETPinhibitor. Theoretically, such an immune response against theexogenously supplied CETP inhibitor could be overcome by administeringincreasing doses of the inhibitor. However, multiple administrations ofdoses of a foreign CETP inhibitor, particularly multiple doses ofever-increasing amounts of such foreign molecules, presents thepossibility of a hypersensitivity reaction, endangering the health ofthe individual being treated. Such problems associated with usingexogenously produced CETP inhibitors are avoided by using thepeptide-based vaccines of this invention, which recruit an individual'sown immune system antibodies to specifically inhibit endogenous CETP.Repeated dosing, graduated dosing, and undesirable side-effects (such asa human anti-mouse antibody (HAMA) response) are avoided by employingthe anti-CETP vaccine approach described herein.

[0086] The CETP vaccine peptide compositions of this invention may beadministered by any route used for vaccination, including: parenterallysuch as intraperitoneally, interperitoneally, intradermally(subcutaneously), intramuscularly, intravenously or orally. Preferably,the vaccines of this invention are administered parenterally, e.g.,intraperitoneally, interperitoneally, intradermally, intramuscularly, orintravenously. If oral administration of a vaccine peptide is desired,any pharmaceutically acceptable oral excipient may be mixed with thevaccine peptides of this invention, for example, such as solutionsapproved for use in the Sabin oral polio vaccine. As with certain othervaccines, such as for tetanus, an occasional booster administration ofthe CETP vaccine peptide compositions may be necessary to maintain adesired level of modulation or inhibition of endogenous CETP. As notedabove, biodegradable microspheres, such as those comprised of poly(DL-lactide-co-glycolide), have been shown to be useful for effectivevaccine delivery and immunization via oral or parenteral routes(Eldridge, J. H., et al., In Immunobiology of Proteins and Peptides V:Vaccines: Mechanisms, Design, and Appications, Atassi M. Z., ed. (PlenumPress, New York, 1989), pp. 191-202).

[0087] Appropriate dosages of the peptide vaccines of this invention areestablished by general vaccine methodologies used in the art based onmeasurable parameters for which the vaccine is proposed to affect,including monitoring for potential contraindications, such ashypersensitivity reaction, erythema, induration, tenderness (see, e.g.,Physician's Desk Reference, 49th Cd., (Medical Economics Data ProductionCo., Mont Vale, N.J., 1995), pp. 1628, 2371 (referring to hepatitis Bvaccine), pp. 1501, 1573, 1575 (referring to measles, mumps, and/orrubella vaccines), pp. 904, 919, 1247, 1257, 1289, 1293, 2363 (referringto diphtheria, tetanus and/or pertussis vaccines)) ; Talwar, G. P., etal., Proc. Natl. Acad Sci. USA, 91: 8532-8536 (1994)). A common andtraditional approach for vaccinating humans is to administer an initialdose of a particular vaccine to sensitize the immune system and thenfollow up by one or more “booster” doses of the vaccine to elicit ananalgesic response by the immune system that was sensitized by theinitial administration of the vaccine (vaccination). Such a “primary andbooster” administration procedure has been well known and commonly usedin the art, as for example, in developing and using measles, polio,tetanus, diphtheria, and hepatitis B vaccines.

[0088] Initially, the amount of a vaccine peptide administered to anindividual may be that required to neutralize the approximate level ofendogenous CETP activity present in the individual prior to vaccination,as can be determined by measuring CETP activity in serum or plasmasamples from the individual for example as determined using acommercially available CETP assay (e.g., Diagnescent Technologies, Inc.,Yonkers, N.Y.). Plasma or serum samples from a vaccinated individual canalso be monitored to determine whether a measurable increase in thelevels of total HDL or HDL-C is seen after administration of the-vaccine peptide using commercially available assays (e.g., availablefrom Wako Chemicals USA, Inc., Richmond, Va.). A rise in theconcentration (titer) of circulating anti-CETP antibodies can bemeasured in plasma or serum samples, for example using an ELISA assay(see, e.g., Example 3). Thus, it is possible and recommended thatinitially it be established whether a rise in anti-CETP antibody can becorrelated with an increase in the level of HDL or HDL-C, or with adecrease in CETP activity. Thereafter, ,one need only monitor a rise intiter of anti-CETP antibody to determine whether a sufficient dosage ofvaccine peptide has been administered or whether a “booster” dose isindicated to elicit an elevated level of anti-CETP antibody. This is thecommon procedure with various established vaccinations, such asvaccination against hepatitis B virus.

[0089] Three-dimensional arterial imaging methods are currentlyavailable which can be used to identify arterial lesions and monitortheir development or regression in an individual (see, for example,McPherson, Scientific American Science & Medicine, pages 22-31,(March/April, 1996)). Thus such imaging methods can be used to monitorthe effectiveness of vaccination with a peptide of this invention.

[0090] A more complete appreciation of this invention and the advantagesthereof can be obtained from the following non-limiting examples.

EXAMPLES Example 1 Design and Synthesis of an Anti-CETP Vaccine Peptide

[0091] To investigate the possibility of eliciting an antibody responseagainst endogenous CETP, a peptide was prepared having a helper T cellepitope portion comprising a universal tetanus toxoid helper T cellepitope and a B cell epitope portion comprising a carboxyl terminalregion of human CETP. A 3 1-amino acid peptide was designed having theamino acid sequence C Q Y I K A N S K F I G I T E F G F P E H L L V D FL Q S L S (SEQ ID NO:2), in which Q Y I K A N S K F I G I T E (aminoacids 2 to 15 of SEQ ID NO:2) is the same amino acid sequence as aminoacids 830 to 843 of the tetanus toxoid protein, F G F P E H L L V D F LQ S L S (amino acids 16 to 31 of SEQ ID NO:2) is the same amino acidsequence as amino acids 461 to 476 SEQ ID NO:4 containing the neutrallipid transfer domain of human CETP and known to be recognized byanti-human CETP Mab TP2 (Wang, S., et al., J. Biol. Chem., 267:17487-17490 (1992); Wang, S., et al., J. Biol. Chem., 268: 1955-1959(1993)), and the amino terminal cysteine (C) residue is present for usein linking the peptide to itself or other molecules if desired. TheCETP-related portion of this synthetic peptide differs from thecorresponding portion of rabbit CETP amino acid sequence only at theglutamic acid (E) residue (see, Nasashima, M., et al., J. Lipid Res.,29:1643-6149 (1988) (cloning of rabbit CETP gene)). However, prior studyhas indicated anti-human CETP Mabs can recognize this correspondingregion of rabbit CETP (see, Hesler, C. B., et al., J. Biol. Chem., 263:5020-5023 (1988)). The peptide was synthesized to order using standardpeptide synthesis methods by Quality Controlled Biochemicals, Inc.(Hopkinton, Mass.).

Example 2 Immunization of Rabbits Against Endogenous CETP

[0092] The synthetic vaccine peptide (SEQ ID NO:2) of Example 1 abovewas injected into New Zealand White Rabbits to test the ability of thevaccine peptide to elicit an immune response against endogenous rabbitCETP. Group I contained three rabbits (rb#1-#3), each of which wassubjected to a protocol for administration of the vaccine peptide. Group11 contained one rabbit (rb#4) as a control that was not treated.

[0093] The general protocol for testing the vaccine peptide in therabbits is shown in FIG. 1. On Day 1, peptide (100 μg) was suspended inthe RIBI™ adjuvant system (RIBI ImmunoChem Research, Inc., Hamilton,Mont.) according to manufacturer's instructions to a final volume of1000 μl and each rabbit of Group I was injected at two intramuscularsites (250 μl per site), subcutaneously at two sites (100 μl per site),and six intradermal sites (50 μl per site). On Day 28, a boost (100 μgof peptide in RIBI™ adjuvant system) was administered as on Day 1. OnDay 56, another boost (100 μg of peptide in RIBI™ adjuvant system) wasadministered as on Day 1.

[0094] Blood samples (approximately 1-5 ml) were withdrawn from the earof each rabbit prior to each initial injection (“prebleed”) and on Days42, 70, and 108, except that there was no pre-bleed for control rabbitrb#4. Blood plasma samples were prepared by standard centrifugationmethods to separate cellular components from the plasma. Plasma sampleswere stored at −70° C. Plasma samples of both Groups I and II wereanalyzed for presence of and increase in titer of anti-CETP antibodiesand for total plasma cholesterol and plasma HDL-C levels.

Example 3 Production of Anti-CETP Antibody in Vaccinated Rabbits

[0095] Direct ELISA for Titering Anti-CETP Antibodies A sandwichenzyme-linked immunosorbent assay (ELISA) was used to titer plasmasamples containing anti-CETP antibody. In this set-up, recombinant humanCETP (human rCETP, obtained from recombinant CHO cell line CHO(AT.)licensed from The Trustees of Columbia University, New York, N.Y.) wasadsorbed to wells of a microtiter dish, and various dilutions of rabbitplasma from the rabbits of Groups I and II were added to each well. Eachwell of a NUNC Maxisorb 96-well plate was coated by overnight exposureat 4° C. to 100 μl of a 1 μg/ml solution of human rCETP in PBS.Non-specific binding was blocked by adding a 1% solution of BSA in PBSand 0.05% Tween to each well and incubating for 2 hours at roomtemperature (20°-22° C.) on a rotating shaker at 150 rpm. The wells werethen washed four times with ELISA wash buffer (PBS+0.05% Tween). Plasmasamples were then diluted 1:10 in dilution buffer (1% BSA in PBS),followed by 6 two-fold serial dilutions in the same buffer. Dilutedsamples (100 μl) were added to the wells, incubated for 2 hours at roomtemperature on a rotating shaker at 150 rpm, and then washed 4 timeswith ELISA wash buffer (PBS+0.05% Tween). To detect bound anti-CETPantibodies, 100 μl of a 1:10,000 dilution of horseradish peroxidase(HRP) labeled goat anti-rabbit immunoglobuln (Southern BiotechnologyAssociates, Inc.; Birmingham, Ala.) in dilution buffer was added, andthe plates were incubated for 2 hours at room temperature on a rotatingshaker at 150 rpm. The wells were then washed four times with ELISA washbuffer (see above), peroxidase substrate TMB (TMB peroxidase substrate,Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md.) added, and theplates were incubated 30 minutes at room temperature. Change in opticaldensity was monitored spectrophotometrically at 450 nm using an ELISAreader (e.g., E-max, Molecular Device Corp., Menlo Park, Calif.). Inthis assay, the O.D. was directly proportional to the amount ofanti-CETP antibodies present in the plasma samples. The resultsindicated that all of the rabbits (rb#1-rb#3) of Group I producedanti-CETP antibody which was specific for recombinant human CETP. Noanti-CETP antibody was produced in the untreated control rabbit (rb#4)of Group I in Example 2. See FIG. 2.

[0096] Competitive ELISA for Detecting Anti-CETP Antibody

[0097] This assay was designed to determine if the vaccinated rabbitshad generated antibodies that bind to the same epitope as the anti-CETPMab TP2 (licensed from The Trustees of Columbia University, New York,N.Y.). A standard competitive ELISA was adapted to detect the presenceof anti-CETP antibodies in rabbit plasma. In this assay, horseradishperoxidase (HRP) was conjugated to the anti-CETP Mab TP2 whichspecifically binds to the 26 amino acid carboxyl terminal fragment ofhuman CETP (Wang et al., J. Biol. Chem., 267: 17487-17490 (1992); Wanget al., J. Biol. Chem., 268: 1955-1959 (1993)).

[0098] The following method was used to conjugate HRP to antibody.Antibody was dialyzed against Na₂CO₃ (50 mM, pH 9.5). The dialyzedantibody was at a concentration of 2 to 5 mg/ml. HRP(Boehringer-Mannheim) was dissolved in sodium acetate buffer (1.0 mM, pH4.4) to a concentration of 6 mg/m The RP was then activated by adding0.2 ml of sodium periodate (21.4 mg/ml acetate buffer, made immediatelybefore use) to every 1 ml of HRP solution, and the activation mixturewas incubated at room temperature on a rocker for 20 minutes. Theactivated HRP was then passed over a G25 column equilibrated withacetate buffer to desalt the activated HRP. An optical density (O.D.) at403 nm corresponds to approximately 1 mg HRP/ml. The desalted, activatedHRP was then added to the dialyzed antibody at an amount equal to onehalf the amount of antibody (by weight, for example, for every 1 mg ofIgG, add 0.5 mg activated HRP), and the mixture was incubated for 2hours at room temperature on a rocker to allow the HRP to conjugate tothe antibody molecules. The conjugation reaction was stopped by adding20 μl of sodium borohydride (10 mg/ml) for every 1 ml of theHRP-antibody conjugation mixture, and the mixture was then incubated onice for 30 minutes. The HRP-conjugated antibody mixture was dialyzedovernight against phosphate buffered saline (PBS) and then centrifuged(Airfuge) for 15 minutes at 30 psi. Thimerosal was added to thesupernatant (HRP-conjugated antibody) to 0.5%, and bovine serum albuminwas added to 1%. The HRP-conjugated antibody preparation was stored at4° C. and protected from light.

[0099] Wells of 96well microtiter plate were coated with CETP byincubating in each well 100 μl of a 300 ng/ml solution of recombinanthuman CETP (obtained from the recombinant CHO cell line CHO(AT),licensed from The Trustees of Columbia University, New York, N.Y.) inphosphate buffered saline (PBS). The wells were drained and the wellswere filled with a 1% (wt/wt) solution of bovine serum albumin (BSA) inPBS and 0.05% (vol./vol.) Tween (Sigma Chemical Co., St. Louis, Mo.) andincubated for 2 hours at room temperature on a rotating shaker(approximately 150 rpm) to block non-specific binding. The wells werewashed four times with ELISA wash buffer (PBS+0.05% Tween), and 100 μlof plasma samples diluted in dilution buffer (1% BSA in PBS) was added.The plates were then incubated for 1 hour at room temperature on arotating shaker as above, and then washed four times with ELISA washbuffer. To each well was next added 100 μl of a 1:100,000 mution ofhorseradish peroxidase-conjugated (labeled) Mab TP2 in dilution buffer.The plates were incubated for 1 hour at room temperature on a rotatingshaker as above, then washed four times with ELISA wash buffer. Thehorseradish peroxidase substrate (e.g., TMB peroxidase substrate,Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md.) was added toeach well and a change in optical density (O.D.) at 450 mm was monitoredspectrophotometrically using an ELISA reader (e.g., E-max, MolecularDevices Corp., Menlo Park, Calif.). In this assay, if antibody wasproduced against the CETP-related portion of the vaccine peptide, suchunlabeled anti-CETP antibody molecules present in the plasma samplescompetes with the labeled TP2 Mab for binding to the CETP adsorbed onthe walls of the wells and an inhibition in color development isobserved as the concentration of plasma sample increases (i.e., O.D. isinversely proportional to the amount of anti-CETP antibody present ineach plasma sample).

[0100] As shown in FIG. 3, such inhibition of TP2 binding to CETP wasobserved in plasma sample from two of the three rabbits that wereadministered the vaccine peptide, thereby indicating production ofCETP-specific antibody (compare graphs of rabbit sera rb#2 and rb#3 withplasma of untreated control rabbit rb#4 in FIG. 3). The strongestinhibition of TP2 binding to CETP was exhibited by plasma of rabbit rb#3(see FIG. 3).

Example 4 Cholesterol and HDL Levels in Plama Samples of VaccinatedRabbits

[0101] The plasma samples taken from rabbits of Groups I and II inExample 2 at various times (days) in the vaccination protocol were alsoassayed for the concentration of total cholesterol (FIG. 4) and HDL-C(FIG. 5). Total plasma cholesterol and HDL-C levels were determinedusing standard commercial assays (Wako Chemicals USA, Inc., Richmond,Va.). The plasma samples of two rabbits (rb#2 and rb#3) that had thehighest anti-CETP antibody titers showed a 2 to 5-fold increase in HDL-Cconcentrations at Day 70 compared to prebleed plasma samples. Rabbits #2and #3 also showed increasing plasma HDL concentrations over timecompared to the control rabbit (rb#4) and the lowest antibody titerrabbit (rb#1) both of which exhibited decreasing HDL concentrations overtime (FIG. 5).

[0102]FIG. 6 shows the ratio of Non-HDL cholesterol (Non-HDL) toHDL-cholesterol (HDL) on Day 70 post-vaccination. The data show a trendtoward an anti-atherogenic profile in the vaccinated rabbit group(hatched) compared to the non-vaccinated (solid) rabbit. Although nosignificant difference in total cholesterol in plasma samples wasobserved, the ratio of Non-HDL/HDL generally declined with a rise inanti-CETP antibody levels in the vaccinated rabbits of Group I.

Example 5 Administration to Transgenic Mice Expressing Human CETP

[0103] A strain of transgenic mice that expresses human CETP hasrecently become commercially available (Biodigm™-CETP mice; PharmakonUSA, Waverly, Pa.). Such mice express human CETP in their livers and arereported to have approximately 50 percent lower levels of HDL-associatedcholesterol than non-transgenic litter mates when fed a normal chowdiet. Such transgenic animals serve as an additional experimental modelto further test vaccine peptides of this invention.

[0104] Two groups consisting of six transgenic CETP-expressing mice wereused to test the same vaccine peptide used in Examples 1 to 4 above.Each mouse of Group I received primary injections of the vaccine peptidedissolved in phosphate buffered saline (PBS) and emulsified withcomplete Freund's adjuvant (1:1) to yield a final concentration of 100μg/100 μl. Each mouse was administered the vaccine peptide mixture in a50 μl dose (50 μg) at each of two subcutaneous sites. On Day 28 andagain on Day 56, the animals were similarly administered boosts of thepeptide vaccine (100 μg) in PBS, except the vaccine peptide wasemulsified with Incomplete Freund's adjuvant. Samples of blood werewithdrawn on Day 42 and Day 63. The mice of control Group II receivedprimary and boost injections of PBS emulsified with adjuvant, butwithout vaccine peptide, in the same manner as the Group I mice. Plasmasamples were prepared as described above for the rabbit plasma samples.

[0105] All Group I mice had significant titers of anti-CETP antibody asmeasured in a wide range of plasma dilutions (1:10 to 1:1,000,000) bydirect ELISA as described above (see FIG. 7). Furthermore, three of thesix mice from Group I were also shown to have anti-CETP antibody thatcompeted with Mab TP2 for binding to recombinant human CETP (as wasfound for rabbits rb#2 and rb#3 in Example 3, above).

Example 6 Immunization of Rabbits Against Endogenous CETP in aCholesterol-Fed Model of Atherosclerosis

[0106] The synthetic vaccine peptide (SEQ ID NO:2) of Example 1 abovewas injected into New Zealand White Rabbits to test the ability of thevaccine peptide to elicit an immune response against endogenous rabbitCETP and to protect or reduce development of atherosclerosis. Group Icontained six rabbits (rb#1-#6), each of which was subjected to aprotocol for administration of the vaccine peptide. Control Group IIcontained six rabbits (rb#7-12) that were vaccinated but not fed a highcholesterol diet. Control Group III contained six rabbits (rb#13-18)that were not vaccinated and not fed a high cholesterol diet. ControlGroup IV contained six rabbits (rb#19-24) that were not vaccinated butfed a high cholesterol diet. The high cholesterol diet was administeredstarting four weeks after a final boost with the vaccine and continuedfor a total of 17 weeks.

[0107] The general protocol for testing the vaccine peptide in therabbits is shown in Table 1 below. On Day 0, peptide (100 μg) wassuspended in the RIBI™ adjuvant system (RIBI ImmunoChem Research, Inc.,Hamilton, Mont.) according to manufacturer's instructions to a finalvolume of 1000 μl, and each rabbit of Group I and II were injected attwo intramuscular sites (250 μl per site), subcutaneously at two sites(100 μl per site), and six intradermal sites (50 μl per site). On Day28, a boost (100 fig of peptide in RIBI™ adjuvant system) wasadministered as on Day 0. On Day 49, another boost (100 μg of peptide inRIBI™ adjuvant system) was administered as on Day 0. On Day 77, Groups Iand IV were fed 0.25% (w/w) cholesterol-enriched diets (rabbit chowsupplemented with cholesterol (Farmer's Exchange, Framingham, Mass.).Groups II and III were fed the same rabbit chow but not supplementedwith cholesterol (Farmer's Exchange, Framingham, Mass.).

[0108] Blood samples (approximately 1-5 ml) were withdrawn from the earof each rabbit prior to each initial injection (“prebleed”) androutinely at approximately every two weeks thereafter. Blood plasmasamples were prepared by standard centrifugation methods to separatecellular components from the plasma. Plasma samples were stored at −70°C. Plasma samples of all Groups were analyzed for presence of andincrease in titer of anti-CETP antibodies and for total plasmacholesterol and plasma HDL-C levels. TABLE 1 RABBIT SCHEDULE procedure,by group CHOW CHOW WEEK 1 2 3 4 group 2 and 3 group 1 and 4 WEEKS/DIETACTUAL DAY −2 B B B B Normal Normal −14 −1 B B B B Normal Normal −6 0B,V B,V B B Normal Normal 0 1 Normal Normal 7 2 B B B B Normal Normal 143 Normal Normal 21 4 B,V B,V B B Normal Normal 28 5 Normal Normal 35 6Normal Normal 42 7 B,V B,V B B Normal Normal 49 8 Normal Normal 56 9Normal Normal 63 10 B B B B Normal Normal 70 11 Transition Transition 077 12 control 0.25% chol 1 84 13 B B B B control 0.25% chol 2 91 14control 0.25% chol 3 98 15 B B B B control 0.25% chol 4 105 16 control0.25% chol 5 112 17 B B B B control 0.25% chol 6 119 18 control 0.25%chol 7 126 19 B B B B control 0.25% chol 8 133 20 control 0.25% chol 9140 21 B B B B control 0.25% chol 10 147 22 control 0.25% chol 11 154 23B B B B control 0.25% chol 12 161 24 control 0.25% chol 13 168 25 B B BB control 0.25% chol 14 175 26 control 0.25% chol 15 182 27 control0.25% chol 16 189 28 B,E B,E control 0.25% chol 17 196

Example 7 Production of Anti-CETP Antibody in Vaccinated Rabbits inAtherosclerotic (Hypercholesterolemia) Model Direct ELISA for TiteringAnti-Recombinant Human CETP Antibodies

[0109] A sandwich enzyme-linked immunosorbent assay (ELISA) was used totiter plasma samples containing anti-CETP antibody. In this set-up,recombinant human CETP (human rCETP, obtained from recombinant CHO cellline CHO(AT) licensed from The Trustees of Columbia University, NewYork, N.Y.) was adsorbed to wells of a microtiter dish, and variousdilutions of rabbit plasma from the rabbits of Groups I - IV were addedto each well. Each well of a NUNC Maxisorb 96-well plate was coated byovernight exposure at 4° C. to 100 μl of a 1 μg/ml solution of humanrCETP in PBS. Non-specific binding was blocked by adding a 1% solutionof BSA in PBS and 0.05% Tween to each well and incubating for 2 hours atroom temperature (20°-22° C.) on a rotating shaker at 150 rpm. The wellswere then washed four times with ELISA wash buffer (PBS+0.05% Tween).Plasma samples were then diluted 1:10 in dilution buffer (1% BSA inPBS), followed by 6 two-fold serial dilutions in the same buffer.Diluted samples (100 μl) were added to the wells, incubated for 2 hoursat room temperature on a rotating shaker at 150 rpm, and then washed 4times with ELISA wash buffer (PBS+0.05% Tween). To detect boundanti-CETP antibodies, 100 μl of a 1:10,000 dilution of horseradishperoxidase (HRP) labeled goat anti-rabbit immunoglobulin (SouthernBiotechnology Associates, Inc.; Birmingham, Ala.) in dilution buffer wasadded, and the plates were incubated for 2 hours at room temperature ona rotating shaker at 150 rpm. The wells were then washed four times withELISA wash buffer (see above), peroxidase substrate TMB (TMB peroxidasesubstrate, Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md.)added, and the plates were incubated 30 minutes at room temperature.Change in optical density was monitored spectrophotometrically at 450 nmusing an ELISA reader (e.g., E-max, Molecular Device Corp., Menlo Park,Calif.). In this assay, the O.D. was directly proportional to the amountof anti-CETP antibodies present in the plasma samples. The resultsindicated that five of the twelve vaccinated rabbits (Groups I and II)produced anti-CETP antibody which was specific for recombinant humanCETP (see FIG. 9). No anti-recombinant human CETP antibody was producedin the untreated control Groups III and IV.

[0110] Direct ELISA for Titering Autoreactive Anti-Rabbit (Endogenous)CETP Antibodies

[0111] A peptide (rabbit peptide) containing an amino acid sequence ofthe endogenous rabbit CETP was synthesized to order using standardpeptide synthesis methods by Quality Controlled Biochemicals, Inc.(Hopkinton, Mass.) having the sequence of SEQ ID NO:7: L Q M D F G F P KH L L V D F L Q S L S, which corresponding to amino acids 457 to 476 ofthe carboxyl terminal region of the human CETP sequence shown in SEQ IDNO:4. This portion of the rabbit CETP amino acid sequence differs fromthe human CETP sequence in that the glutamic acid residue at position465 of the human sequence (SEQ ID NO:4) is replaced with a lysineresidue (see amino acid 9 in SEQ ID NO:7). For the purposes of thisassay the rabbit peptide was purchased as a biotinylated derivative(biotin covalently attached to the amino terminal leucine residue).

[0112] Preblocked streptavidin-coated microtiter plates were prepared asfollows. One hundred μl of 5 μg/ml streptavidin (catalog #43-4302, ZymedLaboratories, Inc., S. San Francisco, Calif.) in PBS was dispensed intoeach well of 96-well microplates with removable strips (catalog#950-2950-00P, LabSystems, Needham, Mass.), sealed and incubated at roomtemperature overnight. Following aspiration of the contents of eachwell, the plates were washed and then blocked with 300 μl of PBScontaining 1% BSA, 5% sucrose, 0.05% Tween 20 and 0.1% gentamicinsulfate, overnight at room temperature. The following day the wells wereemptied and allowed to dry overnight before storing, sealed withdesiccant, at 4° C., until use. Rabbit peptide was then added to eachwell (100 μl of 1 μg/ml solution in PBS (GIBCO BRL) supplemented with10% (w/v) bovine serum albumin (BSA)) and incubated for 1 hour withshaking at 150 rpm at room temperature (22° C.). Plates were washedthree times with Wash Buffer (PBS supplemented with 0.05% Tween-20) toremove unbound peptide. Rabbit plasma samples were diluted (initially1:40, then serially by half thereafter) in PBS supplemented with 5% BSAand 1% gelatin and 100 μl of each dilution were incubated for 90 minutesat room temperature with shaking at 150 rpm. Plates were then washedthree times with Wash Buffer. Goat-anti-rabbit IgG labelled withhorseradish peroxidase (Southern Biotechnology Associates, Inc.,Birmingham, Ala.) was diluted 1:5000 in Wash Buffer and 100 μl added toeach well which were then incubated for 90 minutes at room temperaturewith shaking at 150 rpm. The plates were washed three times with WashBuffer and then incubated with peroxidase substrate TMB (TMB peroxidasesubstrate, Kirkegaard & Perry Laboratories, Inc., Gaithersburg, Md.)added, and the plates were incubated 30 minutes at room temperature.Change in optical density was monitored spectrophotometrically at 450 nmusing an ELISA reader (e.g., E-max, Molecular Device Corp., Menlo Park,Calif.). In this assay, the O.D. was directly proportional to the amountof anti-rabbit CETP antibodies present in the plasma samples.

[0113] The results indicate that at three weeks post final boost (andprior to administering the cholesterol supplemented diet to Groups I andIV) six of the twelve vaccinated animals (three from each of Groups Iand II) produced antibodies that reacted with the peptide derived fromthe endogenous rabbit CETP sequence (See FIGS. 10A and 10B).

[0114] The data showed that the vaccine peptide was capable of elicitingautoreactive antibodies to the rabbit endogenous CETP. This indicatesthat the combination of the tetanus toxoid T cell epitope and the B cellepitope of the carboxyl terminal region of CETP is capable of breakingtolerance to a specific self protein, i.e., in this case, endogenousrabbit CETP.

Example 9 Cholesterol and HDL Levels in Plasma Samples of VaccinatedRabbits in Atherosclerotic Model

[0115] The plasma samples taken from rabbits of Groups in Example 6 atvarious times (days) in the vaccination protocol were also assayed forthe concentration of total cholesterol (FIG. 11) and HDL-C (FIG. 12).Total plasma cholesterol and HDL-C levels were determined using standardcommercial assays (Wako Chemicals USA, Inc., Richmond, Va.).

[0116] The plasma samples of Groups I (vaccinated) and IV(non-vaccinated) showed an increase (hypercholesterolemia) in totalcholesterol due to administration of the cholesterol supplemented diet(FIG. 11). Groups II (vaccinated) and m (non-vaccinated) did not exhibitdiet-induced hypercholesterolemia (FIG. 11).

[0117] Similarly, Groups I and IV fed the cholesterol supplemented dietshowed an increase in HDL-C (FIG. 12). Preliminary analysis of the datafor Group II, indicated that the three animals with the highestanti-CETP antibody titers also showed an increase in their levels ofHDL-C compared to pre-vaccination levels. The preliminary analysis alsoindicated that in three of the five animals in Group III, no significantchange in the levels of HDL-C were observed compared to pre-vaccinationlevels.

Example 10 Measurement of Aortic Atherosclerotic Lesions in Rabbits in aCholesterol-Fed Model of Atherosclerosis

[0118] Aortas were removed from all surviving rabbits (four in Groups Iand IV, five in Group II, six in Group III) after 17 weeks (Day 196 fromprimary vaccination) on cholesterol supplemented diet in Groups I and IVand control diet (no cholesterol supplement) in Groups II and III. Deathof the non-surviving rabbits was shown by necropsy to be due to hairballs and not, therefore, experimental design. Each aorta was opened foran enface view to examine for atherosclerotic lesions. The full lengthof the aorta, i.e., from the aortic arch in the heart to the bifurcationin the lower abdomen, was stained with Oil Red O to detect lesions. Thelesions were measured and the data quantitated using a computer program(The Morphometer, Woods Hole Educational Associated, Woods Hole, Mass.).

[0119] The results in FIG. 13 of this analysis demonstrated astatistically significant reduction in the size of lesions in animals ofGroup I (vaccinated, cholesterol-supplemented diet) as compared to thesize of lesions in animals in Group IV (non-vaccinated,cholesterol-supplemented diet). The results showed that the peptidevaccine was capable of reducing the area of atherosclerotic lesions inanimals fed cholesterol supplemented (hypercholesterolemic) diets bygreater than 50%. Non-vaccinated animals (Group IV) had lesions thatcovered an average of 45% of the total area of the aorta, whereasvaccinated animals (Group I) had lesions that covered an average of 19%of the total area of the aorta.

[0120] Although a number of embodiments have been described above, itwill be understood by those skilled in the art that modifications andvariations of the described compositions and methods may be made withoutdeparting from either the spirit of the invention or the scope of theappended claims. The articles and publications cited herein areincorporated by reference.

1 9 1 26 PRT Artificial Sequence C - terminal 26 amino acids of HumanCETP 1 Arg Asp Gly Phe Leu Leu Leu Gln Met Asp Phe Gly Phe Pro Glu His 15 10 15 Leu Leu Val Asp Phe Leu Gln Ser Leu Ser 20 25 2 31 PRTArtificial Sequence vaccine peptide of the invention 2 Cys Gln Tyr IleLys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Phe 1 5 10 15 Gly Phe ProGlu His Leu Leu Val Asp Phe Leu Gln Ser Leu Ser 20 25 30 3 21 PRTArtificial Sequence helper T cell epitope of tetanus toxin 3 Phe Asn AsnPhe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser 1 5 10 15 Ala SerHis Leu Glu 20 4 476 PRT Homo Sapiens 4 Cys Ser Lys Gly Thr Ser His GluAla Gly Ile Val Cys Arg Ile Thr 1 5 10 15 Lys Pro Ala Leu Leu Val LeuAsn His Glu Thr Ala Lys Val Ile Gln 20 25 30 Thr Ala Phe Gln Arg Ala SerTyr Pro Asp Ile Thr Gly Glu Lys Ala 35 40 45 Met Met Leu Leu Gly Gln ValLys Tyr Gly Leu His Asn Ile Gln Ile 50 55 60 Ser His Leu Ser Ile Ala SerSer Gln Val Glu Leu Val Glu Ala Lys 65 70 75 80 Ser Ile Asp Val Ser IleGln Asn Val Ser Val Val Phe Lys Gly Thr 85 90 95 Leu Lys Tyr Gly Tyr ThrThr Ala Trp Trp Leu Gly Ile Asp Gln Ser 100 105 110 Ile Asp Phe Glu IleAsp Ser Ala Ile Asp Leu Gln Ile Asn Thr Gln 115 120 125 Leu Thr Cys AspSer Gly Arg Val Arg Thr Asp Ala Pro Asp Cys Tyr 130 135 140 Leu Ser PheHis Lys Leu Leu Leu His Leu Gln Gly Glu Arg Glu Pro 145 150 155 160 GlyTrp Ile Lys Gln Leu Phe Thr Asn Phe Ile Ser Phe Thr Leu Lys 165 170 175Leu Val Leu Lys Gly Gln Ile Cys Lys Glu Ile Asn Val Ile Ser Asn 180 185190 Ile Met Ala Asp Phe Val Gln Thr Arg Ala Ala Ser Ile Leu Ser Asp 195200 205 Gly Asp Ile Gly Val Asp Ile Ser Leu Thr Gly Asp Pro Val Ile Thr210 215 220 Ala Ser Tyr Leu Glu Ser His His Lys Gly His Phe Ile Tyr LysAsn 225 230 235 240 Val Ser Glu Asp Leu Pro Leu Pro Thr Phe Ser Pro ThrLeu Leu Gly 245 250 255 Asp Ser Arg Met Leu Tyr Phe Trp Phe Ser Glu ArgVal Phe His Ser 260 265 270 Leu Ala Lys Val Ala Phe Gln Asp Gly Arg LeuMet Leu Ser Leu Met 275 280 285 Gly Asp Glu Phe Lys Ala Val Leu Glu ThrTrp Gly Phe Asn Thr Asn 290 295 300 Gln Glu Ile Phe Gln Glu Val Val GlyGly Phe Pro Ser Gln Ala Gln 305 310 315 320 Val Thr Val His Cys Leu LysMet Pro Lys Ile Ser Cys Gln Asn Lys 325 330 335 Gly Val Val Val Asn SerSer Val Met Val Lys Phe Leu Phe Pro Arg 340 345 350 Pro Asp Gln Gln HisSer Val Ala Tyr Thr Phe Glu Glu Asp Ile Val 355 360 365 Thr Thr Val GlnAla Ser Tyr Ser Lys Lys Lys Leu Phe Leu Ser Leu 370 375 380 Leu Asp PheGln Ile Thr Pro Lys Thr Val Ser Asn Leu Thr Glu Ser 385 390 395 400 SerSer Glu Ser Ile Gln Ser Phe Leu Gln Ser Met Ile Thr Ala Val 405 410 415Gly Ile Pro Glu Val Met Ser Arg Leu Glu Val Val Phe Thr Ala Leu 420 425430 Met Asn Ser Lys Gly Val Ser Leu Phe Asp Ile Ile Asn Pro Glu Ile 435440 445 Ile Thr Arg Asp Gly Phe Leu Leu Leu Gln Met Asp Phe Gly Phe Pro450 455 460 Glu His Leu Leu Val Asp Phe Leu Gln Ser Leu Ser 465 470 4755 1428 DNA Homo Sapiens 5 tgctccaaag gcacctcgca cgaggcaggc atcgtgtgccgcatcaccaa gcctgccctc 60 ctggtgttga accacgagac tgccaaggtg atccagaccgccttccagcg agccagctac 120 ccagatatca cgggcgagaa ggccatgatg ctccttggccaagtcaagta tgggttgcac 180 aacatccaga tcagccactt gtccatcgcc agcagccaggtggagctggt ggaagccaag 240 tccattgatg tctccattca gaacgtgtct gtggtcttcaaggggaccct gaagtatggc 300 tacaccactg cctggtggct gggtattgat cagtccattgacttcgagat cgactctgcc 360 attgacctcc agatcaacac acagctgacc tgtgactctggtagagtgcg gaccgatgcc 420 cctgactgct acctgtcttt ccataagctg ctcctgcatctccaagggga gcgagagcct 480 gggtggatca agcagctgtt cacaaatttc atctccttcaccctgaagct ggtcctgaag 540 ggacagatct gcaaagagat caacgtcatc tctaacatcatggccgattt tgtccagaca 600 agggctgcca gcatcctttc agatggagac attggggtggacatttccct gacaggtgat 660 cccgtcatca cagcctccta cctggagtcc catcacaagggtcatttcat ctacaagaat 720 gtctcagagg acctccccct ccccaccttc tcgcccacactgctggggga ctcccgcatg 780 ctgtacttct ggttctctga gcgagtcttc cactcgctggccaaggtagc tttccaggat 840 ggccgcctca tgctcagcct gatgggagac gagttcaaggcagtgctgga gacctggggc 900 ttcaacacca accaggaaat cttccaagag gttgtcggcggcttccccag ccaggcccaa 960 gtcaccgtcc actgcctcaa gatgcccaag atctcctgccaaaacaaggg agtcgtggtc 1020 aattcttcag tgatggtgaa attcctcttt ccacgcccagaccagcaaca ttctgtagct 1080 tacacatttg aagaggatat cgtgactacc gtccaggcctcctattctaa gaaaaagctc 1140 ttcttaagcc tcttggattt ccagattaca ccaaagactgtttccaactt gactgagagc 1200 agctccgagt ccatccagag cttcctgcag tcaatgatcaccgctgtggg catccctgag 1260 gtcatgtctc ggctcgaggt agtgtttaca gccctcatgaacagcaaagg cgtgagcctc 1320 ttcgacatca tcaaccctga gattatcact cgagatggcttcctgctgct gcagatggac 1380 tttggcttcc ctgagcacct gctggtggat ttcctccagagcttgagc 1428 6 496 PRT rabbit 6 Cys Pro Lys Gly Ala Ser Tyr Glu Ala GlyIle Val Cys Arg Ile Thr 1 5 10 15 Lys Pro Ala Leu Leu Val Leu Asn GlnGlu Thr Ala Lys Val Val Gln 20 25 30 Thr Ala Phe Gln Arg Ala Gly Tyr ProAsp Val Ser Gly Glu Arg Ala 35 40 45 Val Met Leu Leu Gly Arg Val Lys TyrGly Leu His Asn Leu Gln Ile 50 55 60 Ser His Leu Ser Ile Ala Ser Ser GlnVal Glu Leu Val Asp Ala Lys 65 70 75 80 Thr Ile Asp Val Ala Ile Gln AsnVal Ser Val Val Phe Lys Gly Thr 85 90 95 Leu Asn Tyr Ser Tyr Thr Ser AlaTrp Gly Leu Gly Ile Asn Gln Ser 100 105 110 Val Asp Phe Glu Ile Asp SerAla Ile Asp Leu Gln Ile Asn Thr Glu 115 120 125 Leu Thr Cys Asp Ala GlySer Val Arg Thr Asn Ala Pro Asp Cys Tyr 130 135 140 Leu Ala Phe His LysLeu Leu Leu His Leu Gln Gly Glu Arg Glu Pro 145 150 155 160 Gly Trp LeuLys Gln Leu Phe Thr Asn Phe Ile Ser Phe Thr Leu Lys 165 170 175 Leu IleLeu Lys Arg Gln Val Cys Asn Glu Ile Asn Thr Ile Ser Asn 180 185 190 IleMet Ala Asp Phe Val Gln Thr Arg Ala Ala Ser Ile Leu Ser Asp 195 200 205Gly Asp Ile Gly Val Asp Ile Ser Val Thr Gly Ala Pro Val Ile Thr 210 215220 Ala Thr Tyr Leu Glu Ser His His Lys Gly His Phe Thr His Lys Asn 225230 235 240 Val Ser Glu Ala Phe Pro Leu Arg Ala Phe Pro Pro Gly Leu LeuGly 245 250 255 Asp Ser Arg Met Leu Tyr Phe Trp Phe Ser Asp Gln Val LeuAsn Ser 260 265 270 Leu Ala Arg Ala Ala Phe Gln Glu Gly Arg Leu Val LeuSer Leu Thr 275 280 285 Gly Asp Glu Phe Lys Lys Val Leu Glu Thr Gln GlyPhe Asp Thr Asn 290 295 300 Gln Glu Ile Phe Gln Glu Leu Ser Arg Gly LeuPro Thr Gly Gln Ala 305 310 315 320 Gln Val Ala Val His Cys Leu Lys ValPro Lys Ile Ser Cys Gln Asn 325 330 335 Arg Gly Val Val Val Ser Ser SerVal Ala Val Thr Phe Arg Phe Pro 340 345 350 Arg Pro Asp Gly Arg Glu AlaVal Ala Tyr Arg Phe Glu Glu Asp Ile 355 360 365 Ile Thr Thr Val Gln AlaSer Tyr Ser Gln Lys Lys Leu Phe Leu His 370 375 380 Leu Leu Asp Phe GlnCys Val Pro Ala Ser Gly Arg Ala Gly Ser Ser 385 390 395 400 Ala Asn LeuSer Val Ala Leu Arg Thr Glu Ala Lys Ala Val Ser Asn 405 410 415 Leu ThrGlu Ser Arg Ser Glu Ser Leu Gln Ser Ser Leu Arg Ser Leu 420 425 430 IleAla Thr Val Gly Ile Pro Glu Val Met Ser Arg Leu Glu Val Ala 435 440 445Phe Thr Ala Leu Met Asn Ser Lys Gly Leu Asp Leu Phe Glu Ile Ile 450 455460 Asn Pro Glu Ile Ile Thr Leu Asp Gly Cys Leu Leu Leu Gln Met Asp 465470 475 480 Phe Gly Phe Pro Lys His Leu Leu Val Asp Phe Leu Gln Ser LeuSer 485 490 495 7 1488 DNA rabbit 7 tgtcccaaag gcgcctccta cgaggctggcatcgtgtgtc gcatcaccaa gcccgccctc 60 ttggtgttga accaagagac ggccaaggtggtccagacgg ccttccagcg cgccggctat 120 ccggacgtca gcggcgagag ggccgtgatgctcctcggcc gggtcaagta cgggctgcac 180 aacctccaga tcagccacct gtccatcgccagcagccagg tggagctggt ggacgccaag 240 accatcgacg tcgccatcca gaacgtgtccgtggtcttca aggggaccct gaactacagc 300 tacacgagtg cctgggggtt gggcatcaatcagtctgtcg acttcgagat cgactctgcc 360 attgacctcc agatcaacac agagctgacctgcgacgctg gcagtgtgcg caccaatgcc 420 cccgactgct acctggcttt ccataaactgctcctgcacc tccaggggga gcgcgagccg 480 gggtggctca agcagctctt cacaaacttcatctccttca ccctgaagct gattctgaag 540 cgacaggtct gcaatgagat caacaccatctccaacatca tggctgactt tgtccagacg 600 agggccgcca gcatcctctc agatggagacatcggggtgg acatttccgt gacgggggcc 660 cctgtcatca cagccaccta cctggagtcccatcacaagg gtcacttcac gcacaagaac 720 gtctccgagg ccttccccct ccgcgccttcccgcccggtc ttctggggga ctcccgcatg 780 ctctacttct ggttctccga tcaagtgctcaactccctgg ccagggccgc cttccaggag 840 ggccgtctcg tgctcagcct gacaggggatgagttcaaga aagtgctgga gacccagggt 900 ttcgacacca accaggaaat cttccaggagctttccagag gccttcccac cggccaggcc 960 caggtagccg tccactgcct taaggtgcccaagatctcct gccagaaccg gggtgtcgtg 1020 gtgtcttctt ccgtcgccgt gacgttccgcttcccccgcc cagatggccg agaagctgtg 1080 gcctacaggt ttgaggagga tatcatcaccaccgtccagg cctcctactc ccagaaaaag 1140 ctcttcctac acctcttgga tttccagtgcgtgccggcca gcggaagggc aggcagctca 1200 gcaaatctct ccgtggccct caggactgaggctaaggctg tttccaacct gactgagagc 1260 cgctccgagt ccctgcagag ctctctccgctccctgatcg ccacggtggg catcccggag 1320 gtcatgtctc ggctcgaggt ggcgttcacagccctcatga acagcaaagg cctggacctc 1380 ttcgaaatca tcaaccccga gattatcactctcgatggct gcctgctgct gcagatggac 1440 ttcggttttc ccaagcacct gctggtggatttcctgcaga gcctgagc 1488 8 50 PRT Artificial Sequence vaccine peptide ofthe invention 8 Cys Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile ThrGlu Leu 1 5 10 15 Phe Pro Arg Pro Asp Gln Gln His Ser Val Ala Tyr ThrPhe Glu Glu 20 25 30 Asp Ile Phe Gly Phe Pro Glu His Leu Leu Val Asp PheLeu Gln Ser 35 40 45 Leu Ser 50 9 50 PRT Artificial Sequence vaccinepeptide of the invention 9 Met Gln Tyr Ile Lys Ala Asn Ser Lys Phe IleGly Ile Thr Glu Arg 1 5 10 15 Phe Pro Arg Pro Asp Gly Arg Glu Ala ValAla Tyr Arg Phe Glu Glu 20 25 30 Asp Ile Phe Gly Phe Pro Lys His Leu LeuVal Asp Phe Leu Gln Ser 35 40 45 Leu Ser 50

1. An isolated peptide comprising a helper T cell epitope portion and aB cell epitope portion, wherein said helper T cell epitope portioncomprises a broad range helper T cell epitope and said B cell epitopeportion comprises a B cell epitope of CETP.
 2. The isolated peptideaccording to claim 1 wherein said B cell epitope portion comprises aportion of human CETP consisting of at least 6 consecutive amino acidsof SEQ ID NO:4 and which are recognized by B cells or antibodies.
 3. Theisolated peptide according to claim 1 wherein said B cell epitope ofCETP is selected from the group consisting of the amino acid sequencedefined by amino acids 349 to 367 of SEQ ID NO:4, amino acids 461 to 476of SEQ ID NO:4; amino acid sequences identified by antigenic epitopeidentifying algorithms, a region involved in neutral lipid binding, aregion involved in neutral lipid transfer activity.
 4. The isolatedpeptide according to claim 1 wherein the helper T cell epitope portioncomprises a helper T cell epitope derived from an antigenic peptideselected from the group consisting of tetanus toxoid, diphtheria toxoid,pertussis vaccine, Bacile Calmette-Guerin (BCG), polio vaccine, measlesvaccine, mumps vaccine, rubella vaccine, purified protein derivative oftuberculin, keyhole limpet hemocyanin, hsp70, and combinations thereof.5. The isolated peptide according to claim 2 wherein the B cell epitopeportion is a peptide consisting of between six and 26 consecutive aminoacids of the carboxyl terminal 26 amino acids of human CETP (SEQ ID NO:1).
 6. The isolated peptide according to claim 2, wherein said B cellepitope portion is a derivative of CETP having neutral lipid binding orneutral lipid transfer actvity.
 7. The isolated peptide according toclaim 1 having an amino terminal cysteine residue.
 8. The isolatedpeptide according to claim 1 comprising the amino acid sequence of SEQID NO:2.
 9. A vaccine comprising a vaccine peptide, said vaccine peptidecomprising a universal helper T cell epitope portion linked to a B cellepitope portion, said B cell epitope portion comprising a B cell epitopeof a CETP.
 10. The vaccine according to claim 9 wherein the T cellepitope portion of the vaccine peptide comprises a helper T cell epitopederived from an antigenic peptide selected from the group consisting oftetanus toxoid, diphtheria toxoid, pertussis vaccine, BacileCalmette-Guerin (BCG), polio vaccine, measles vaccine, mumps vaccine,rubella vaccine, purified protein derivative of tuberculin keyholelimpet hemocyanin, hsp70, and combinations thereof.
 11. The vaccineaccording to claim 9 wherein the helper T cell epitope portion of thevaccine peptide comprises a helper T cell epitope selected from thegroup consisting of the amino acid sequence of amino acids 830 to 843 oftetanus toxin protein (amino acids 2 to 15 of SEQ ID NO:2), the aminoacid sequence of amino acids 947 to 967 of tetanus toxin protein (SEQ IDNO:3), and combinations thereof.
 12. The vaccine according to claim 9wherein the CETP is human CETP.
 13. The vaccine according to claim 12wherein the B cell epitope portion of said vaccine peptide comprises a Bcell epitope of human CETP selected from the group consisting of betweensix and 26 consecutive amino acids of the carboxyl terminal 26 aminoacids of human CETP (SEQ ID NO:1).
 14. The vaccine according to claim 9wherein the vaccine peptide further comprises an amino terminal cysteineresidue.
 15. The vaccine according to claim 9 further comprising avaccine peptide covalently linked to multiple copies of complementprotein C3d.
 16. The vaccine according to claim 9 further comprising avaccine peptide derivatized with carbohydrate structures which becomecovalently linked in vivo with complement protein C3d.
 17. A method ofelevating the ratio of circulating HDL to circulating LDL, VLDL, ortotal cholesterol in a human or other animal comprising administering tothe human or animal a vaccine composition comprising a vaccine peptidecomprising a helper T cell epitope portion and a B cell epitope portion,wherein said B cell epitope portion comprises a B cell epitope of a CETPof said human or other animal.
 18. The method according to claim 17wherein said B cell epitope portion comprises a carboxyl terminal regionof CETP involved in neutral lipid binding or neutral lipid transferactivity.
 19. The method according to claim 17 wherein the helper T cellepitope portion of the vaccine peptide comprises a T cell epitopeselected from the group consisting of the amino acid sequence of aminoacids 830 to 843 of tetanus toxin protein (amino acids 2 to 16 of SEQ IDNO:2) and the amino acid sequence of amino acids 947 to 967 of tetanustoxin protein of SEQ ID NO:3.
 20. The method according to claim 17wherein the B cell epitope portion of the vaccine peptide is selectedfrom the group consisting of between six and 26 consecutive amino acidsof SEQ ID NO:
 1. 21. The method according to claim 17 wherein thevaccine peptide further comprises an amino terminal cysteine residue.22. A method of decreasing the level of endogenous CETP activity in ahuman or other animal comprising administering to the human or animal avaccine peptide comprising a helper T cell epitope portion linked to a Bcell epitope portion comprising a B cell epitope of a CETP of a human oranimal.
 23. The method according to claim 22 wherein the vaccine peptideis administered in an amount sufficient to elicit production in saidhuman or other animal of anti-CETP antibodies.
 24. A method of alterringthe catabolism of HDL-cholesterol to decrease the development ofatherosclerotic lesions in a human or other animal comprisingadministering to the human or animal a vaccine peptide comprising ahelper T cell epitope portion linked to a B cell epitope portion, saidhelper T cell epitope portion comprising a broad range T cell epitopeand said B cell epitope portion comprising a B cell epitope of CETP. 25.A method of increasing the level of circulating HDL in a human or otheranimal comprising administering to the human or animal a vaccine peptidecomprising a helper T cell epitope portion and a B cell epitope portion,wherein said B cell epitope portion comprises a B cell epitope of a CETPof said human or other animal.
 26. The method according to claim 25,wherein the helper T cell epitope portion comprises a helper T cellepitope derived from an antigenic peptide selected from the groupconsisting of tetanus toxoid, diphtheria toxoid, pertussis vaccine,Bacile Calmette-Guerin (BCG), polio vaccine, measles vaccine, mumpsvaccine, rubella vaccine, purified protein derivative of tuberculin,keyhole limpet hemocyanin, and combinations thereof.
 27. The methodaccording to claim 25, wherein the B cell epitope portion comprises acarboxyl terminal region of human CETP consisting of between six and 26consecutive amino acids of SEQ ID NO:
 1. 28. A method fortherapeutically or prophylactically treating atherosclerosis in a humanor other animal in need of treatment thereof comprising administering tosaid human or other animal a vaccine peptide in a pharmaceuticallyacceptable buffer, said vaccine peptide comprising a helper T cellepitope portion comprising a helper T cell epitope and a B cell epitopeportion comprising a B cell epitope of CETP.
 29. The method for treatingatherosclerosis according to claim 28 wherein said helper T cell epitopeportion comprises a helper T cell epitope derived from an antigenicpeptide selected from the group consisting of tetanus toxoid, diphtheriatoxoid, pertussis vaccine, Bacile Calmette-Guerin (BCG), polio vaccine,measles vaccine, mumps vaccine, rubella vaccine, purified proteinderivative of tuberculin keyhole limpet hemocyanin, hsp70, andcombinations thereof.
 30. A method of making an anti-CETP vaccine tomodulate endogenous CETP activity or to treat therapeutically orprophylactically atherosclerosis, comprising: selecting a B cell epitopeof CETP wherein said B cell epitope does not include a majorhistocompatibility complex class I T cell epitope; selecting a helper Tcell epitope derived from an antigenic peptide which is not derived fromCETP; and linking said B cell epitope of CETP and said helper T cellepitope to form an immunogenic moiety.
 31. The method according to claim30 wherein said B cell epitope portion is covalently linked to saidhelper T cell epitope.
 32. The method according to claim 30, whereinsaid B cell epitope portion is covalently linked to said helper T cellepitope via a covalent bond selected from the group consisting ofpeptide bonds and disulfide bonds.
 33. The method according to claim 30wherein said B cell epitope portion is linked to said helper T cellepitope via a cross-linker molecule.
 34. The method according to claim30 wherein said B cell epitope portion is linked to said helper T cellepitope via a bridge of amino acids.
 35. The method according to claim30 wherein said B cell epitope portion and said helper T cell epitopeare linked to a common carrier molecule.
 36. The method according toclaim 30 wherein said B cell epitope portion is linked to said helper Tcell epitope to form a vaccine peptide and further comprising the stepof linking said vaccine peptide to a common carrier molecule.